Genomic relationships among Nicotiana species with different ploidy levels revealed by 5S rDNA spacer sequences and FISH/GISH

We used the intergenic spacer sequences of the 5S ribosomal RNA genes (5S rDNA) to obtain insights into the genomic origin of putative amphidiploid/tetraploid species with 2n = 48 and their descendants in Nicotiana. Amplification of the spacer sequences and subsequent multiple alignment using the consensus sequences from each species, showed that two Australian species shared common large deletions, suggesting that the origin of the 5S rDNA is closely related in these species. Comparison of the spacer sequences with those from diploid (2n = 24) Nicotiana species made it possible to detect some groups consisting of the sequences from the 2n = 24 and 2n = 48 level species. Chromosomal localizations of the 5S rDNA arrays were similar in most groups. The relationships suggested by the 5S rDNA were also assessed at the genome level by using genomic in situ hybridization. We showed that the grouping based on the 5S rDNA spacer sequence reflects high genomic homology between 2n = 24 and 2n = 48 level species. As a result, the putative polyploid species such as N. debneyi, N. quadrivalvis, and N. africana were suggested to involve the close relatives of the diploid species such as N. glauca, N. obtusifolia and N. sylvestris, and N. langsdorffii, respectively, in their speciation. Our results are generally in agreement with the relationships previously suggested by morphological and cytogenetic observations, and some novel relationships were also revealed.     

Nicotiana cybrids with Petunia chloroplasts

Summary Protoplasts of a chloroplast-defective cultivar of Nicotiana tabacum were fused with gamma-irradiated protoplasts of Petunia hybrida. Over 100 photoautotrophic plants were regenerated; of these 94 were tested for Petunia chloroplast traits and all but one had Petunia chloroplasts based on their sensitivity to the fungal toxin, tentoxin. Chloroplast DNA was analysed for 3 of the sensitive plants and was shown to be identical to Petunia chloroplast DNA. Most of the plants (about 70%) appeared to be normal N. tabacum plants, based on morphology and chromosome number. They were fully fertile with normal pollen viability, seed set, and seed viability. The remaining 30% of the plants showed varying degrees of vegetative and reproductive abnormalities.The techniques of somatic cell genetics have led to many possible nuclear-organellar combinations that may be considered as cybrids. In this paper, we use the term to include the combination of nucleus from one species and chloroplast from another species     

Seven of eight species in Nicotiana section Suaveolentes have common factors leading to hybrid lethality in crosses with Nicotiana tabacum

Background and Aims

Reproductive isolation is a mechanism that separates species, and is classified into two types: prezygotic and postzygotic. Inviability of hybrids, or hybrid lethality, is a type of postzygotic isolation and is observed in some plant species, including Nicotiana species. Previous work has shown that the Q chromosome, which belongs to the S subgenome of N. tabacum, encodes one or more genes leading to hybrid lethality in some crosses.

Methods

Interspecific crosses of eight wild species were conducted in section Suaveolentes (which consists of species restricted to Australasia and Africa) with the cultivated species Nicotiana tabacum. Hybrid seedlings were cultivated at 28, 34 or 36 °C, and PCR and chromosome analysis were performed.

Results and Conclusions

Seven of eight wild species produced inviable hybrids after crossing. Hybrid lethality, which was observed in all crosses at 28 °C, was Type II lethality, with the characteristic symptoms of browning of hypocotyl and roots; lethality was suppressed at elevated temperatures (34 or 36 °C). Furthermore, one or more genes on the Q chromosome of N. tabacum were absolutely responsible for hybrid lethality, suggesting that many species of section Suaveolentes share the same factor that triggers hybrid lethality by interaction with the genes on the Q chromosome. Exceptionally, only one wild species, N. fragrans, produced 100 % viable hybrids after crossing with N. tabacum, suggesting that N. fragrans has no factor triggering hybrid lethality.     

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.     

Peroxidases in the genus Nicotiana

Summary Leaf peroxidases of 60 Nicotiana species, 19 cultivars, autopolyploids, interspecific hybrids, and amphidiploids have been compared by polyacrylamide gel block electrophoresis. At least 19 peroxidase bands, four cathodic and 15 anodic, were detected in the species which varied from two bands in N. arentsii to 12 bands in N. tabacum. Tihe cultivars of the latter species failed to reveal any intraspecific variation. Specific difference and varietal resemblance in root peroxidase bands were also observed in nine species and 20 varieties analyzed. Zymograms from autopolyploids and amphidiploids appeared to be identical to that of diploid parents, suggesting that peroxidase banding patterns are independent of ploidy levels. An additive manner of parental peroxidase bands without hybrid enzyme formation in interspecific hybrids and the failure of dissociating peroxidases into subunits lead to a hypothesis that peroxidases in Nicotiana may be controlled by multiple, dominant genes and/or codominant alleles in chromosomes of different genomes. This is in keeping with the lack of relationship between ploidy level and peroxidase banding pattern. Also, species with different chromosome numbers shared many peroxidases in common that possibly reflects a residual homology of peroxidase loci among Nicotiana species. Some species classified in different sections or subgenera but having a common geographic center of origin, showed close similarities in peroxidase zymogram. Results suggest that these species may be closely related in phylogeny, and/or geographic isolation changes the peroxidase genes through mutation and selection. Based on leaf peroxidase zymograms of F ₁ hybrids, a putative ancestor of N. tomentosiformis was the progenitor of N. tabacum at its inception.

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Zusammenfassung Die Peroxidasen in Blättern von 60 Nicotiana-Spezies, 19 Sorten und einer Anzahl Autopolyploiden, interspezifischen Hybriden und Amphidiploiden wurden mittels Polyacrylamid-Gelblockelektrophorese verglichen. In den Species wurden mindestens 19 Peroxidasebanden festgestellt (4 kathodische und 15 anodische) mit einem Variationsbereich von 2 Banden bei N. arentsii bis 12 Banden bei N. tabacum. Sorten der letztgenannten Spezies ließen keine intraspezifische Variation entdecken. Spezifische Unterschiede und eine Sortenähnlichkeit der Wurzelperoxydase-Banden wurden auch in den untersuchten 9 Species und 20 Sorten beobachtet. Zymogramme von Autopolyploiden und Amphidiploiden schienen identisch zu sein mit denen der diploiden Eltern und lassen darauf schließen, daß die Muster der Peroxidasebanden von der Ploidiestufe unabhängig sind. Eine weitere Art elterlicher Peroxidasebanden ohne Hybrid-Enzymbildung bei interspezifischen Hybriden und die Unmöglichkeit der Trennung der Peroxidasen in Untereinheiten führen zu der Hypothese, daß die Peroxidasen bei Nicotiana durch multiple dominante Gene und/oder co-dominante Allele in den Chromosomen verschiedener Genome gesteuert werden. Das paßt gut zu der fehlenden Beziehung zwischen Ploidiestufe und Peroxidasebandenmuster. Auch Spezies mit verschiedenen Chromosomenzahlen hatten viele Peroxidasen gemeinsam, was möglicherweise auf eine Rest-Homologie der Peroxidase-Loci bei den Nicotiana-Spezies schließen läßt. Deutliche Ähnlichkeiten im Peroxidase-Zymogramm zeigten einige Spezies, die zwar in verschiedene Sektionen oder Subgenera eingestuft sind, aber ein gemeinsames geographisches Ursprungszentrum haben. Die Ergebnisse lassen vermuten, daß sich diese Spezies phylogenetisch nahestehen und/oder die geographische Isolation die Peroxidasegene durch Mutation und Selektion verändert hat. Nach dem Blatt-Peroxidase-Zymogramm von F₁-Hybriden war ein vermutlicher Vorfahr von N. tomentosiformis der Ahn von N. tabacum.

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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.     

Use of transferable Nicotiana tabacum L. microsatellite markers for investigating genetic diversity in the genus Nicotiana.

The recent development of microsatellite markers for tobacco, Nicotiana tabacum L., may be valuable for genetic studies within the genus Nicotiana. The first objective was to evaluate transferability of 100 N. tabacum microsatellite primer combinations to 5 diploid species closely related to tobacco. The number of primer combinations that amplified scorable bands in these species ranged from 42 to 56. Additional objectives were to assess levels of genetic diversity amongst available accessions of diploid relatives closely related to tobacco (species of sections Sylvestres and Tomentosae), and to evaluate the efficacy of microsatellite markers for establishing species relationships in comparison with existing phylogenetic reconstructions. A subset of 46 primer combinations was therefore used to genotype 3 synthetic tobaccos and an expanded collection of 51 Nicotiana accessions representing 15 species. The average genetic similarity for 7 diverse accessions of tobacco was greater than the average similarity for N. otophora accessions, but lower than the average genetic similarities for N. sylvestris, N. tomentosa, N. kawakamii, and N. tomentosiformis accessions. A microsatellite-based phylogenetic tree was largely congruent with taxonomic representations based on morphological, cytological, and molecular observations. Results will be useful for selection of parents for creation of diploid mapping populations and for germplasm introgression activities.     

Production of Nicotiana tabacum x Nicotiana acuminata hybrid by ovule culture

Using a conventional sexual crossing technique, Nicotiana tabacum x N. acuminata was not produced. After the fertilized ovules were cultured for 20 days in a liquid Nitsch H medium, germination was observed. The roots grew rapidly but leaves did not. However, plantlets were produced in an H medium containing Benzyladenine or Kinetin (0.01–0.1 mg/l). The plantlets grew and flowered in a greenhouse. The chromosome number of the hybrid was 36 and its morphological characteristics were intermediate between those of parental species.Abbreviations BA Benzyl adenine - K Kinetin     

In vitro regeneration of Nicotiana africana plants from explants of different type and mesophyll protoplasts

Methods of in vitro cultivation of an African endemic species Nicotiana africana (Solanaceae) possessing some valuable traits have been elaborated. Influence of different concentrations of auxin (alpha-naphtaleneacetic acid) and cytokinins (6-benzylaminopurine, zeatin, thidiazuron) on morphogenesis and plant regeneration has been analysed using leaves and internodes as explants. The optimum method of regeneration of N. africana shoots in leaf explants is cultivation in the presence of 0.1 mg/l NAA and 1 mg/l BA; in internode explants--cultivation on the medium containing 0.5 mg/l NAA and 1 mg/l zeatin. The use of thidiazuron was the most effective at the concentration of 0.4 mg/l with subsequent transfer of explants to hormone-free medium. Method of isolation and culture of mesophyll protoplasts enabling to regenerate N. africana plants during 3-4 months has been proposed.     

Comparison of Nicotiana tabacum and Nicotiana nesophila hybrids produced by ovule culture and protoplast fusion

Summary Somatic hybrids were produced between Nicotiana tabacum and N. nesophila, two species incapable of conventional sexual hybridization. Sexual hybrids, though, could be produced between these two species by using ovule culture only when N. nesophila was female. Clones of somatic hybrids were compared with sexual hybrids. Statistically significant variation was observed between clones, but not between sexual hybrids, for pollen viability, flower morphology, leaf morphology, and trichome density. As all clones of somatic hybrids have 96 chromosomes, the variability could not be explained by interclonal variation in chromosome number. Variation between somatic hybrids could be the result of cytoplasmic segregation or recombination, mitotic recombination or small chromosomal rearrangements prior to plant regeneration. Variation between clones could be exploited as these interspecies hybrids are now being used to incorporate disease resistance into cultivated tobacco.     

Modulation of flowering responses in different Nicotiana varieties

We have identified and characterized a FLOWERING PROMOTING FACTOR 1(FPF1) gene from tobacco (NtFPF1). Over-expression of NtFPF1 leads to early flowering in the day-neutral tobacco Nicotiana tabacum cv. Hicks, and under inductive photoperiods also in the short-day Nicotiana tabacum cv. Hicks Maryland Mammoth (MM) tobacco and the long-day plant Nicotiana sylvestris. N. sylvestris wild-type plants remained in the rosette stage and never flowered under non-inductive short-days, whereas 35S::NtFPF1 transgenic plants bolted but did not flower. However, if treated with gibberellins, transgenic N. sylvestrisplants flowered much faster under non-inductive short days than corresponding wild type plants, indicating an additive effect of gibberellins and the NtFPF1 protein in flowering time control. The day-neutral wild type cv. Hicks and the short-day cv. Hicks MM plants exhibit an initial rosette stage, both under short- and long-days. In the transgenic lines, this rosette stage was completely abolished. Wild-type plants of cv. Hicks MM never flowered under long days; however, all transgenic lines over-expressing NtFPF1 flowered under this otherwise non-inductive photoperiod.     

In vitro hybridization in an incompatible cross Nicotiana glutinosa X Nicotiana megalosiphon

Summary Using conventional techniques interspecific hybridization between N. glutinosa (2n=24) and N. megalosiphon (2n=40) results in failure while the reciprocal is successful. Attempts were made here to obtain an F₁ hybrid of N. glutinosa X N. megalosiphon by embryo rescue. These reciprocal hybrids resembled each other in their phenotypic and chromosomal behaviour, i.e. there is broad spectrum of phenotypic variation coupled to chromosomal range from 2n-28 to 32. This may be due to the functional disharmony between chromosome sets of two unrelated species resulting in the elimination of chromosomes.     

Ribosomal RNA cistron number in Nicotiana species and derived haploids

Twelve Nicotiana species, and haploids obtained by anther culture from three of these species, were assayed for the proportion of DNA complementary to ribosomal RNA. No variation was observed in the proportion of DNA coding for ribosomal RNA between diploid and haploid plants within a given species. Wide variation was observed in this proportion between the species with values ranging between 0.068% and 0.43% of the total DNA. No relationship between the proportion of DNA complementary to rRNA and the chromosome number of the species was observed.     

Distribution of the Tnt1 retrotransposon family in the amphidiploid tobacco (Nicotiana tabacum) and its wild Nicotiana relatives

Transposable elements can generate considerable genetic diversity. Here we examine the distribution of the Tnt1 retrotransposon family in representative species of the genus Nicotiana . We show that multiple Tnt1 insertions are found in all Nicotiana species. However, Tnt1 insertions are too polymorphic to reveal species relationships. This indicates that Tnt1 has amplified rapidly and independently after Nicotiana speciation. We compare patterns of Tnt1 insertion in allotetraploid tobacco ( N. tabacum ) with those in the diploid species that are most closely related to the progenitors of tobacco, N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). We found no evidence for Tnt1 insertion sites of N. otophora origin in tobacco. Nicotiana sylvestris has a higher Tnt1 content than N. tomentosiformis and the elements are distributed more uniformly across the genome. This is reflected in tobacco where there is a higher Tnt1 content in S-genome chromosomes. However, the total Tnt1 content of tobacco is not the sum of the two modern-day parental species. We also observed tobacco-specific Tnt1 insertions and an absence of tobacco Tnt1 insertion sites in the diploid relatives. These data indicate Tnt1 evolution subsequent to allopolyploidy. We explore the possibility that fast evolution of Tnt1 is associated with 'genomic-shock' arising out of interspecific hybridization and allopolyploidy.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 639–649.     

Nuclear DNA codes for Nicotiana ferredoxin.

Ferredoxin was purified from 10 species of Nicotiana and spinach leaves. Fingerprints showed all to contain five major tryptic peptides. Some of the spinach peptides were different in RF and mobility from the Nicotiana peptides, but none of the Nicotiana ferredoxins had peptides which could distinguish one species of ferredoxin from another. Electrofocusing S-carbaminomethylcysteinyl ferredoxins showed spinach ferredoxin to have a more acidic and N. glutinosa ferredoxin a slightly more acidic isoelectric point than the other 9 Nicotiana species which were alike. Electro-focusing ferredoxin from the hybrid N. glutinosa female times N. glauca male resolved two bands or isozymes of ferredoxin, one corresponding to N. glutinosa, the other to N. glauca, the code for the latter having come from the DNA in the N. glauca pollen used to form the hybrid plant. N. glutinosa ferredoxin does not contain methionine and is different from N. tabacum and N. glauca ferredoxins which contain methionine. The N. glutinosa female times N. glauca male ferredoxin contained one-half the methionine found in N. glauca ferredoxin, thus confirming that some of the genetic information for ferredoxin in the hybrid was originally contained in the nuclear DNA of N. glauca.     

Study of the divergence of moderately repetitive sequences in Nicotiana species and in protoclones of Nicotiana plumbaginifolia Viviani

Summary Molecular DNA markers can be very useful to assess the amount of genetic variation and are thus important for taxonomic studies. Two moderately repetitive sequences were isolated from N. plumbaginifolia leaf DNA and used to screen various Nicotiana species. A huge variability was detected among species belonging to the same subgenus or the same section, which could be utilized for a molecular taxonomy of the genus Nicotiana. Although variation at the DNA level between somaclonal lines was reported, we did not find evidence for variability of both repetitive sequences in established callus culture obtained from protoplasts of Nicotiana plumbaginifolia.