Seven of eight wild species produced inviable hybrids after crossing. Hybrid lethality, which was observed in all crosses at 28 degrees C, was Type II lethality, with the characteristic symptoms of browning of hypocotyl and roots; lethality was suppressed at elevated temperatures (34 or 36 degrees 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.

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Hybrid seedlings from the cross Nicotiana tabacum × N. suaveolens, obtained by test-tube pollination and ovule culture, expressed lethality at 28°C. Characteristic lethal symptoms in these hybrid seedlings consisted of browning of hypocotyls and roots. One hundred and seventeen hybrid seedlings were eventually obtained by the use of test-tube pollination and ovule culture. Hybrid seedlings maintained at 36°C did not express any lethal symptoms. Hybrid seedlings used for further experiments were transferred to 36°C immediately after germination at 28°C. When hybrid seedlings cultured at 36°C were transferred to 28°C, their growth stopped and lethal symptoms were expressed. During the progressive expression of lethality, apoptotic features such as chromatin condensation, nuclear fragmentation and DNA fragmentation were detected. On the other hand, there was no sign of apoptotic cell death in the hybrid seedlings at 36°C. Based on the observation that the same lethal symptoms and the same apoptotic features were observed in the reciprocal cross, N. suaveolens × N. tabacum, we suggest that not only the underlying causes of hybrid lethality but also the underlying causes of apoptotic cell death are due to the interaction of coexisting heterogeneous genomes, rather than to the effect of cytoplasmic genes. Furthermore, the progression of apoptotic cell death in the cross N. tabacum × N. suaveolens began in stems and roots, followed by leaves.

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Hybrid seedlings from the cross Nicotiana tabacum x N. suaveolens express lethality at 28 degrees 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 degrees C. To suppress hybrid lethality, hybrid seedlings obtained were transferred to 36 degrees 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 degrees C were transferred to 28 degrees 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.

Hybrid lethality expressed in the interspecific hybrid of Nicotiana suaveolens Lehm. x 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 degrees C but not under high-temperature conditions (36 degrees 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.

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 x N. sylvestris, but not in hybrid seedlings from the cross N. debneyi x 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 x N. debneyi.

Hybrid weakness is a reproductive barrier that is found in many plant species. In rice, the hybrid weakness caused by two complementary genes, Hwc1 and Hwc2, has been surveyed intensively. However, their gene products and the molecular mechanism that causes hybrid weakness have remained unknown. We performed linkage analyses of Hwc1, narrowed down the area of interest to 60 kb, and identified eight candidate genes. In the F(2) population, in which both Hwc1 and Hwc2 genes were segregated, plants were separable into four classes according to their respective phenotypes: severe type, semi-severe type, F(1) type, and normal type. Severe type plants show such severe symptoms that they could produce only tiny shoot-like structures; they were unable to generate roots. Genetic analyses using closely linked DNA markers of the two genes showed that the symptoms of the F(2) plants were explainable by the genotypes of Hwc1 and Hwc2. Weakness was observed in plants that have both Hwc1 and Hwc2. In Hwc1 homozygote, the symptoms worsened and severe type or semi-severe type plants appeared. Consequently, Hwc1 should have a gene dosage effect and be a semi-dominant gene. The dosage effect of Hwc2 was recognizable, but it was not so severe as that in Hwc1. These results are useful to elucidate the mechanism that causes the hybrid weakness phenomenon and the role of each causal gene in hybrid weakness.