Hybrid lethality is one of the sexual barriers preventing wide hybridization. In the genus Nicotiana, hybrid lethality is observed in some interspecific cross combinations. Interspesific hybridization of Nicotiana stocktonii×N. tabacum has not been carried out from the viewpoint of expression of lethality. N. tabacum has an S subgenome derived from N. sylvestris and a T subgenome derived from N. tomentosiformis. In this study, hybrid seedlings from the cross N. stocktonii×N. tabacum, were obtained by ovule culture. The hybrid seedlings were classified as normal seedlings, tumorous types and vitrified plants, and they did not express lethality. Hybrid seedlings from the cross N. stocktonii×N. sylvestris expressed lethality. Moreover, hybrid seedlings from N. stocktonii×N. tomentosiformis also expressed lethality. This suggests that both the S genome of N. sylvestris and the T genome of N. tomentosiformis have factors causing lethality in hybrids with N. stocktonii. However, hybrid seedlings from N. stocktonii×N. tabacum did not express lethal symptoms in this study. We suggested that the factors responsible for lethality in N. tabacum must have been lost or no longer expressed during the process of speciation, probably due to reorganization and modification of the genomes.

Monosomic lines of Nicotiana tabacum are helpful to confirm the location of genes on specific chromosomes. In the cross N. nudicaulis and N. tabacum, hybrid seedlings express lethal symptoms, which are controlled by the S subgenome of N. tabacum. To identify the responsible chromosome, we needed to produce chromosome lacking lines (CLLs) of N. tabacum L. "Red Russian" and use them to cross with N. nudicaulis. From a cross of (N. tabacum × N. tomentosiformis) × N. tabacum, 380 BC1 individuals were obtained. Using a Haplo-Q line (a monosomic line lacking the single linkage group 11) and N. tabacum, we found that qPCR is a simple and reliable screening method for CLLs of N. tabacum. The marker PT30342 is located on linkage group 11, and the ∆Ct value (Ct Actin -Ct PT30342) was 2.0 for a disomic line and was 1.097 for a Haplo-Q line. By the use of flow cytometry, qPCR and chromosome counting together as a screening method, we identified 6 CLLs lacking 2 to 6 chromosomes. Compared with conventional methods, our method is a rapid technique for making and screening CLLs ofthe S or S/T subgenome of N. tabacum. Further, these CLLs will be useful to identify the location of two or more factors on chromosomes controlling a variety of genetic problems affecting breeding. Here, we only made CLLs of the S or S/T subgenome of N. tabacum. We will use the method established in this study to produce CLLs of the T subgenome of N. tabacum, and gather a full set of CLLs of N. tabacum. qPCR could also be applied to the identification of chromosome aberrations in other plants.