This study sought to elucidate the mechanism by which individual hybrid plants overcome hybrid lethality, whether spontaneously or by artificially propagation. To achieve this goal, we formulated and tested two (non-exclusive) working hypotheses based on previous findings regarding the lethality-overcoming phenomenon of products of a N. suaveolens x N. tabacum cross. Hypothesis 1 was that seedlings that overcome lethality in the N. suaveolens x N. tabacum cross occur by formation of male gametes lacking the lethality-associated Nt6549g30 gene, which is lost as a result of reciprocal translocations, between homoeologous chromosomes, that occur during meiosis in N. tabacum. Hypothesis 2 focused on the observation that culturing of N. suaveolens x N. tabacum seedlings in the presence of cytokinin yields regenerated plants that overcome hybrid lethality at high frequency; we proposed that hybrid lethality is overcome as a result of the appearance of cells lacking the distal part of the Q chromosome during culturing.
To examine Hypothesis 1, we first identified 16 surviving seedlings from 12,943 germinated N. suaveolens x N. tabacum seedling individuals. The results of RAPD-PCR and morphological observation demonstrated that some of the viable seedlings were indeed hybrids (possessing genomes from both parents) and showed extremely low expression levels of immune response-related genes, confirming these to be hybrid seedlings that had overcome lethality (Figs. 1, 2, 3, Tables 1, S1). Specifically, 11 hybrid seedlings that had overcome lethality were obtained, and the frequency of appearance of hybrid seedlings that overcame lethality in this cross combination was calculated to be 8.5 x 10-4. This value was close to 1.1 x 10-3, which is the frequency of appearance of hybrid seedlings that overcame lethality in a previous study of the N. tabacum x N. africana cross18.
PCR results showed that 5 of 8 of the lethality-overcoming seedlings lacked the distal part of the Q chromosome, a region that contains Nt6549g30 (Table 1). When qPCR was performed using two sets of primer pairs designed to amplify DNA at the end of the Q' chromosome, the copy number of the amplified region from 4 of the 5 individuals that overcame lethality was approximately twice that of seedlings that exhibited lethality (Fig. 6). Therefore, in these 4 individuals, it appears that the end of the Q chromosome, including Nt6549g30, was replaced due to a reciprocal translocation between the Q chromosome and its homoeologous chromosome (Q'). These data support the existence of a lethality-overcoming mechanism consistent with our Hypothesis 1. Figure 7 shows a suggested model, based on our results, for the lethality-overcoming mechanism in N. suaveolens x N. tabacum. In this model, a hybrid seedling that overcomes lethality is produced by fertilization of a female gamete from N. suaveolens by a male gamete from N. tabacum; notably, the male gamete has lost Nt6549g30 due to reciprocal translocation, during meiosis, between the distal regions of the Q and Q' chromosomes. This model is consistent with the overcoming of lethality in N. tabacum x N. africana18 and in other interspecific crosses within the genus Nicotiana where N. tabacum serves as one parent.
GRAS-Di analysis of one hybrid (s17-2) that escaped lethality suggested that lethality was overcome by reciprocal translocation. Notably, an amplicon derived from the region extending to Q chromosomal DNA bp 81360673 was detected, but no amplicon derived from the DNA region beyond bp 81497158 was detected (Table S4). This result implied that the break-point of the reciprocal translocation detected in s17-2 is located within a region of about 136 kb, positioned between bp 81360673 and bp 81497158 on the Q chromosome.
For one (s17-1) of the five seedlings that overcame lethality, qPCR analysis showed that the copy number of the amplified region was the same as that of the lethal seedlings (Fig. 6). This observation implicated deletion of the distal part of the Q chromosome in overcoming lethality in this individual. Specifically, for s17-1, products that would be amplified by three primer pairs (PT52864, PT55075, and PT60178) were not detected; these fragments would span the SSR marker located near the center of the Q chromosome (at 62.25 cM) (Table 1). Therefore, reciprocal translocations between homoeologous chromosomes may be more likely to occur in the region near the end of the Q chromosome, while deletions of relevant loci may be more likely to occur in the region closer to the center of the chromosome. In heteroploid synthetic Brassica plants, both chromosomal deletions and reciprocal translocations (between homoeologous chromosomes) have been reported in synthetic progeny29-30. We postulate that the deletion at the distal part of the Q chromosome may be the result of aberrant segregation and cleavage of chromosomes following synapsis between homoeologous chromosomes during meiosis of N. tabacum, which is a heteroploid.
In 3 individuals (s14-1, s14-2, and s14-4) that overcame lethality, a number that represented approximately 38% of the 8 seedlings that overcame lethality and were subjected to PCR, amplicons were detected for all of the primer pairs (Table 1). Therefore, it appeared that these individuals did not lack the lethality-associated gene on the N. tabacum side (Nt6549g30) as a result of reciprocal translocation or deletion of the distal part of the Q chromosome end. In N. tabacum x N. africana, it has been suggested that about 37% of lethality-overcoming seedlings have intact Q chromosomes18. Both N. suaveolens and N. africana are included in the Suaveolentes section and are thought to be derived from amphidiploid progenitors31. Based on these results, we hypothesized that reciprocal translocation or deletion of the chromosome segment on which the lethality-associated gene is located also can occur in N. suaveolens and N. africana. Overcoming lethality in the seedlings of N. suaveolens x N. tabacum and N. tabacum x N. africana, in which no reciprocal translocation or deletion of the distal part of the Q chromosome was detected, may result from chromosomal mutations due to synapsis of homoeologous chromosomes. However, since the lethality-associated genes in N. suaveolens and N. africana are unknown and the genomic sequences of those species remain unpublished, this hypothesis cannot be tested at this time.
To examine our Hypothesis 2, seedlings of N. suaveolens x N. tabacum were cultured in a medium containing a high concentration of cytokinin, in an effort to obtain viable regenerated plants. The results of RAPD-PCR and morphological observation suggested the resulting regenerated plants had genomes from both parents, confirming that these individuals were indeed hybrids. These plants also showed strongly decreased expression levels of immune response-related genes. Therefore, we judged these individuals to be regenerated hybrid plants that had overcome lethality (Figs. 2, 3, 4, Tables 1, S1). In 3 (r18-1, r18-7, and r19-3) of the 12 regenerated plants that overcame lethality, PCR analysis showed that the distal part of the Q chromosome (the region containing Nt6549g30) had been lost, as seen for some of the seedlings that overcame lethality (Table 1). Additionally, qPCR of these three individuals showed that the copy number of the amplified region was the same as that of lethal seedlings (Fig. 6). These results suggested that the deletion of the distal part of the Q chromosome in these three individuals contributed to overcoming lethality, an inference consistent with our Hypothesis 2. Hypothesis 2 was based on the previous finding that ROS are generated in culture26 and are involved in chromosomal cleavage27. In the future, it will be necessary to verify that ROS are involved in the deletion of the distal part of the Q chromosome. Such confirmatory experiments will require demonstrating ROS production in hybrid seedlings that are cultured in a medium containing a high concentration of cytokinin, and then showing that inhibition of ROS production prevents the emergence of viable shoots.
In 9 of the 12 regenerated plants that overcame lethality, PCR analysis showed that amplicons were detected for all primer pairs (Table 1). Thus, in these individuals, it appears that lethality was overcome by a mechanism other than the deletion of the distal part of the Q chromosome. In addition to recombination-associated events that result in chromosomal lesions, mutation by DNA methylation and by DNA base substitution and deletion is known to occur in plant tissue culture26. It also has been suggested that the supplementation of medium with high-concentration BAP induces mutation32. Furthermore, certain R genes have been reported to show frequent DNA base substitutions and deletions during mitosis33. It is possible that these various genetic mutations occur in the lethality-associated genes of N. suaveolens and N. tabacum, resulting in the formation of viable shoots during tissue culture. It also is possible that ROS production under culture conditions induces deletion of the chromosomal region containing the lethality-associated gene in the N. suaveolens genome. To clarify these mutational mechanisms, it will be necessary to perform detailed sequence analysis of the Nt6549g30 locus in regenerated plants that overcome lethality, and to identify the lethality-associated chromosome and gene in the N. suaveolens genome.
In the present study, expression analysis of immune-response-related genes (e.g., PAL1, PRB1-like, LOX1, and PDF-like protein 1) was performed in the process of detecting seedlings and regenerated plants that overcame lethality. In A. thaliana, the PAL1 gene product is involved in the synthesis of salicylic acid34, and PRB1 encodes a PR-1-like protein expressed in response to ethylene and methyl jasmonate35. On the other hand, the LOX1 gene product has been implicated in jasmonic acid synthesis36, and PDF1.2 encodes a jasmonic acid-responsive defensin36. In the present work, the expression levels of these genes in seedlings and regenerated plants that overcame lethality were much lower than those in the lethal seedlings at 6 DAG (Fig. 3). These data strongly suggested that the seedlings and regenerated plants that overcome lethality lack the function of the lethality-associated gene that triggers the immune-response signal, a deficiency that presumably is due to some genetic variation.
Hybrid lethality is a type of reproductive isolation and a mechanism for maintaining species independence. On the other hand, the results of the present study suggest that chromosomal instability permits hybrids to overcome hybrid lethality, thereby escaping reproductive isolation. We postulate that this phenomenon may contribute to the birth of new species in the process of plant evolution. In addition, hybrid lethality has been reported in a wide range of crop species9 and is a serious obstacle in hybrid breeding of crops. The findings obtained in this study may lead to a technique for artificially breaking reproductive isolation by inducing chromosomal instability.
In summary, we found that the loss of the distal part of the Q chromosome is involved in overcoming hybrid lethality in seedlings and in regenerated plants obtained by culturing hybrid seedlings produced by interspecific Nicotiana crosses. We propose that loss of this factor is the result of reciprocal translocation and deletion between the Q chromosome and its homoeologous partner chromosome (Q') in hybrid seedlings, and of a distal deletion in the Q chromosome in regenerated plants. These findings, which relate to the mechanism of breaking reproductive isolation, are likely to be important in the evolution of polyploid plant species and in the expansion of genetic resources available for cross-breeding.