Hybrid lethality in interspecific hybrids between Nicotiana tabacum and N. suaveolens: evidence that the Q chromosome causes hybrid lethality based on Q-chromosome-specific DNA markers

Hybrid seedlings from the cross Nicotiana tabacum × N. suaveolens express lethality at 28°C. We carried out a cross between monosomic lines of N. tabacum lacking the Q chromosome and N. suaveolens by test-tube pollination and ovule culture at 28°C. To suppress hybrid lethality, hybrid seedlings obtained were transferred to 36°C immediately after germination and cultured. We determined whether Q-chromosome-specific DNA markers were detected among hybrid seedlings. When hybrid seedlings cultured at 36°C were transferred to 28°C, hybrid seedlings in which Q-chromosome-specific DNA markers were detected expressed hybrid lethality, while hybrid seedlings in which Q-chromosome-specific DNA markers were not detected did not express hybrid lethality. From these results, we concluded that the presence of the Q chromosome of N. tabacum is related to hybrid lethality observed in crosses between N. tabacum and N. suaveolens. This is the first report that clearly demonstrates the relationship between a certain chromosome and hybrid lethality in the genus Nicotiana using chromosome-specific DNA markers. Additionally, we confirmed that the Q chromosome belongs to the S subgenome because Q-chromosome-specific DNA markers were detected only in N. sylvestris.     

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.     

Species Origin of Genomic Factors in Nicotiana nudicaulis Watson Controlling Hybrid Lethality in Interspecific Hybrids between N. nudicaulis Watson and N. tabacum L

Hybrid lethality is expressed at 28°C in the cross Nicotiana nudicaulis×N. tabacum. The S subgenome of N. tabacum has been identified as controlling this hybrid lethality. To clarify the responsible genomic factor(s) of N. nudicaulis, we crossed N. trigonophylla (paternal progenitor of N. nudicaulis) with N. tabacum, because hybrids between N. sylvestris (maternal progenitor of N. nudicaulis) and N. tabacum are viable when grown in a greenhouse. In the cross N. trigonophylla×N. tabacum, approximately 50% of hybrids were vitrified, 20% were viable, and 20% were nonviable at 28°C. To reveal which subgenome of N. tabacum was responsible for these phenotypes, we crossed N. trigonophylla with two progenitors of N. tabacum, N. sylvestris (SS) and N. tomentosiformis (TT). In the cross N. sylvestris×N. trigonophylla, we confirmed that over half of hybrids of N. sylvestris×N. trigonophylla were vitrified, and none of the hybrids of N. trigonophylla×N. tomentosiformis were. The results imply that the S subgenome, encoding a gene or genes inducing hybrid lethality in the cross between N. nudicaulis and N. tabacum, has one or more genomic factors that induce vitrification. Furthermore, in vitrified hybrids of N. trigonophylla×N. tabacum and N. sylvestris×N. trigonophylla, we found that nuclear fragmentation, which progresses during expression of hybrid lethality, was accompanied by vitrification. This observation suggests that vitrification has a relationship to hybrid lethality. Based on these results, we speculate that when N. nudicaulis was formed approximately 5 million years ago, several causative genomic factors determining phenotypes of hybrid seedlings were inherited from N. trigonophylla. Subsequently, genome downsizing and various recombination-based processes took place. Some of the causative genomic factors were lost and some became genomic factor(s) controlling hybrid lethality in extant N. nudicaulis.     

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.     

Genes in S and T subgenomes are responsible for hybrid lethality in interspecific hybrids between Nicotiana tabacum and Nicotiana occidentalis

Background

Many species of Nicotiana section Suaveolentes produce inviable F₁ hybrids after crossing with Nicotiana tabacum (genome constitution SSTT), a phenomenon that is often called hybrid lethality. Through crosses with monosomic lines of N. tabacum lacking a Q chromosome, we previously determined that hybrid lethality is caused by interaction between gene(s) on the Q chromosome belonging to the S subgenome of N. tabacum and gene(s) in Suaveolentes species. Here, we examined if hybrid seedlings from the cross N. occidentalis (section Suaveolentes)×N. tabacum are inviable despite a lack of the Q chromosome.

Methodology/Principal Findings

Hybrid lethality in the cross of N. occidentalis×N. tabacum was characterized by shoots with fading color. This symptom differed from what has been previously observed in lethal crosses between many species in section Suaveolentes and N. tabacum. In crosses of monosomic N. tabacum plants lacking the Q chromosome with N. occidentalis, hybrid lethality was observed in hybrid seedlings either lacking or possessing the Q chromosome. N. occidentalis was then crossed with two progenitors of N. tabacum, N. sylvestris (SS) and N. tomentosiformis (TT), to reveal which subgenome of N. tabacum contains gene(s) responsible for hybrid lethality. Hybrid seedlings from the crosses N. occidentalis×N. tomentosiformis and N. occidentalis×N. sylvestris were inviable.

Conclusions/Significance

Although the specific symptoms of hybrid lethality in the cross N. occidentalis×N. tabacum were similar to those appearing in hybrids from the cross N. occidentalis×N. tomentosiformis, genes in both the S and T subgenomes of N. tabacum appear responsible for hybrid lethality in crosses with N. occidentalis.     

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.     

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.     

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.     

Apoptotic cell death induces temperature-sensitive lethality in hybrid seedlings and calli derived from the cross of Nicotiana suaveolens×N. tabacum

Hybrid lethality expressed in the interspecific hybrid of Nicotiana suaveolens Lehm. ×N. tabacum L. cv. Hicks-2 is one of the mechanisms for reproductive isolation and it is temperature-sensitive. Apoptotic changes were detected in the cells of hybrid seedlings and calli expressing lethality at 28 °C but not under high-temperature conditions (36 °C), when the lethality is suppressed. Condensation of chromatin, fragmentation of nuclei and cytoplasmic reduction are the cytological changes associated with apoptosis leading to hybrid lethality. Fragmentation of nuclei was correlated with the lethal symptoms in both hybrid seedlings and calli, as confirmed by fluorimetry of the nuclear DNA using laser scanning cytometry. Agarose gel analysis of DNA extracted from hybrid seedlings and calli showing lethal symptoms revealed a specific ladder pattern suggesting nucleosomal fragmentation which is one of the biochemical changes of apoptosis. In-situ detection using terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) showed that this process occurred in distinct stages on each organ of hybrid seedlings and centripetally in hybrid calli. From these results, we confirmed that cell death inducing hybrid lethality was indeed apoptosis. Received: 23 December 1999 / Accepted: 5 April 2000     

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     

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     

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.     

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.     

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.     

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.     

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.     

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.     

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     

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.