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.


Introduction
Reproductive isolation involves various types of prezygotic and postzygotic barriers in plants [1]. Although these barriers contribute to the formation of species, they are obstacles for plant breeders, especially in breeding programs involving wide hybridization. Hybrid plants from normal parents often show weak growth or death before maturity. Several terms are used to describe this phenomenon, including hybrid lethality, hybrid weakness, hybrid necrosis, and hybrid inviability. Hybrid lethality is a type of postzygotic barrier and is observed in certain cross combinations in numerous plant species, including Nicotiana sp. [2], Oryza sp. [3,4], Phaseolus vulgaris [5], Lactuca sp. [6], and Arabidopsis thaliana [7].
The genus Nicotiana comprises 76 species that are predominantly distributed in the Americas and Australia [8]. This complex genus has attracted the attention of many researchers investigating evolutionary processes. The most well-studied and characterized species in this genus is cultivated tobacco, Nicotiana tabacum (2n = 48, SSTT), which belongs to section Nicotiana. N. tabacum is a natural allotetraploid (amphidiploid) that originated by interspecific hybridization of N. sylvestris (2n = 24, SS) with N. tomentosiformis (2n = 24, TT) and subsequent chromosome doubling [9][10][11][12][13][14][15][16]. Each chromosome of N. tabacum is lettered alphabetically (A-Z, excluding X and Y); chromosomes A-L and M-Z belong to the T and S subgenomes, respectively. A complete set of 24 monosomic lines of N. tabacum (Haplo-A-Z), which lack a certain chromosome, has been established in the genetic background of 'Red Russian' and is useful for locating genes on specific chromosomes [17][18][19]. Monosomic analyses have identified one N. tabacum chromosome containing a gene or genes that trigger hybrid lethality [20][21][22].
Hybrid lethality in the genus Nicotiana is currently classified into four types based on surface symptoms, as follows: Type I, browning of shoot apex and root tips; Type II, browning of hypocotyl and roots; Type III, yellowing of true leaves; and Type IV, formation of multiple shoots [2]. Although several methods to overcome hybrid lethality have been reported in the genus Nicotiana, their effectiveness is dependent on the type of hybrid lethality encountered. For example, hybrid lethality of Types I, II, and III is temperature sensitive; i.e., hybrid lethality is observed at 28uC, but not at elevated temperatures of approximately 36uC. In contrast, Type IV lethality is not suppressed at elevated temperatures [2].
Our previous studies indicate that many species in Nicotiana section Suaveolentes produce inviable hybrids after crosses with N. tabacum [20][21][22]. Section Suaveolentes includes 26 species, most of which are endemic to Australia, although three species are found in other locations. For example, N. debneyi is distributed in Eastern Australia, New Caledonia, and Lord Howe Island, while N. fragrans is distributed in New Caledonia, and the Loyalty, Tonga, and Marquesas Islands [23,24]. One species, N. africana, is distributed in Namibia (South-West Africa), and represents the only Nicotiana species discovered in Africa to date [25]. Species in section Suaveolentes are geographically isolated from the majority of species in other sections, which are distributed in the Americas.
All species in section Suaveolentes are allotetraploids and comprise an almost complete aneuploid series of n = 16-24, with only n = 17 yet to be identified. Section Suaveolentes is considered to have originated from a single polyploid event approximately 10 mya, followed by speciation to produce the species known today [26]. This hypothesis is supported by recent studies indicating that section Suaveolentes is a monophyletic group based on analyses of internal transcribed spacer (ITS) regions [14], plastid genes [27], and nuclear-encoded chloroplast-expressed glutamine synthetase (ncpGS) [16]. Furthermore, progenitors of this section have been identified based on sequence analyses of ncpGS; the maternal progenitor is N. sylvestris and the paternal progenitor is section Trigonophyllae [16]. One group of Suaveolentes species is genetically distant from N. tabacum, even though they share the same maternal progenitor, N. sylvestris [14,16,27]. Although Suaveolentes species are valuable as sources of disease resistance [28][29][30] and cytoplasmic male sterility [31,32], the occurrence of hybrid lethality represents a barrier for introducing desirable characteristics into N. tabacum by interspecific crosses.
After crosses with N. tabacum, nine species in section Suaveolentes, namely N. africana, N. debneyi, N. excelsior, N. goodspeedii, N. gossei, N. maritima, N. megalosiphon, N. suaveolens, and N. velutina, produce inviable hybrids, whereas only N. benthamiana and N. fragrans generate 100% viable hybrids [20][21][22]33]. Hybrid lethality observed in all crosses at 28uC is Type II and is suppressed at temperatures of 34-36uC. We also previously conducted genetic analyses of hybrid lethality, which revealed that the Q chromosome in the S subgenome of N. tabacum encodes one or more genes leading to hybrid lethality in crosses using the nine species of section Suaveolentes [20][21][22]. Based on the existence of a single origin of section Suaveolentes, we proposed that many species in section Suaveolentes share the same gene(s) triggering hybrid lethality by interaction with gene(s) on the Q chromosome [22].
In the present study, we examined hybrid lethality in crosses between N. occidentalis, which belongs to section Suaveolentes, and N. tabacum. N. occidentalis is closely related to other Australian species in section Suaveolentes and is distant from N. tabacum based on recently constructed phylogenetic trees [14,16,27]. Although Ternovskii et al. [34] reported that hybrid seedlings from the cross N. occidentalis R6N. tabacum = die without forming roots at the cotyledonary stage, the nature of the lethality was incompletely described. Here, based on phenotypic and genetic analyses, we demonstrate that N. occidentalis exhibits a distinct type of hybrid lethality after crosses with N. tabacum than that observed with the nine Suaveolentes species examined in our previous studies.

Results
Temperature-sensitive lethality in hybrid seedlings of N. occidentalis R6N. tabacum = Reciprocal crosses between N. occidentalis and N. tabacum (cultivars 'Red Russian' and 'Samsun NN') were carried out using conventional cross-pollination (Table 1). When N. occidentalis was used as the paternal parent, flowers of N. tabacum dropped approximately 7 days after pollination. The ovaries and ovules of these flowers did not enlarge, suggesting that fertilization did not occur. In contrast, when N. occidentalis was used as the maternal parent, seeds that germinated well at 28uC under in-vitro conditions were obtained.
Hybrid seedlings from the cross N. occidentalis R6N. tabacum = were grown in vitro at 24, 28, and 34uC ( Table 2). At 24uC, hybrid seedlings grew normally and developed cotyledons and roots by approximately 3 days after germination (DAG), although root growth stopped at this stage ( Figure 1A). The color of cotyledons and the hypocotyl began to fade approximately 8 DAG ( Figure 1B, C), with hybrid seedlings dying at about 30 DAG ( Figure 1D). A characteristic symptom of hybrid lethality was the fading of shoot color. As this symptom was distinct from those of the four known types of Nicotiana hybrid lethality [2], the type of lethality observed here was designated as Type V.
Hybrid seedlings also displayed Type V lethality at 28uC, although the lethal symptoms appeared later (approximately 30 DAG) than for seedlings grown at 24uC, with the seedlings surviving for a few months ( Table 2). In contrast, hybrid seedlings grown at 34uC did not show lethal symptoms for at least 60 DAG ( Table 2). When these hybrid seedlings were transferred to 28uC, they ceased growing and died 40-70 days after the transfer. The three parental species, N. occidentalis, 'Red Russian', and 'Samsun NN', grew normally without the appearance of lethal symptoms for at least 60 DAG at 24, 28, and 34uC (Table 2, Figure 1E-J).
Hybrid seedlings die even when they lack the Q chromosome N. tabacum monosomic plants lacking the Q chromosome, namely F 1 progeny derived from the cross N. tabacum Haplo-Q R6'Samsun NN' = [35], were crossed with N. occidentalis to determine whether the Q chromosome is responsible for hybrid lethality. Monosomic plants were used as maternal parents, as the transmission of the monosomic condition through pollen seldom occurs in N. tabacum [36]. When N. tabacum was pollinated with N. occidentalis, fertilization did not occur, as described above. Therefore, test-tube pollination and ovule culture were performed to overcome this crossing barrier.
Twenty-nine placentas of monosomic plants were pollinated in vitro, resulting in 445 enlarged ovules that were cultured at 28uC. The 30 hybrid seedlings obtained from the cultured ovules were further grown at 34uC and then assessed for the presence or absence of the Q chromosome using three Q-chromosome-specific sequence-tagged-site (STS) markers: QCS2, QCS3, and QCS4 [20,35]. Five of the hybrid seedlings exhibited abnormal morphology and lacked cotyledons and leaves. Because genomic DNA could not be isolated from these five seedlings, they were excluded from further analyses.
Using the Q-chromosome-specific STS markers, the remaining 25 hybrid seedlings were divided into two types: 11 hybrid seedlings possessing the Q chromosome and 14 hybrid seedlings lacking the Q chromosome (Table 3). Both types of hybrid seedlings were cultured for 30 DAG and then transferred from 34 to 28uC. After 40-70 days, both types of seedlings showed lethal symptoms and died ( Figure 1K, L). Although a characteristic symptom of hybrid lethality is the fading of shoot color, as reported above, no difference in this symptom was observed between the two types of hybrid seedlings. These results indicated that lack of the Q chromosome does not suppress hybrid lethality in N. tabacum R6N. occidentalis = progeny.
Causal genes for hybrid lethality exist in both the S and T subgenomes of N. tabacum Two progenitors of N. tabacum, N. sylvestris and N. tomentosiformis, were crossed with N. occidentalis to reveal which subgenome of N. tabacum encodes gene(s) responsible for hybrid lethality. Conventional cross-pollination was successful when N. occidentalis was used as the maternal parent (Table 4). Seeds from two crosses, as well as those of N. sylvestris and N. tomentosiformis, germinated well at 28uC under in-vitro conditions. Hybrid seedlings and the parental species were grown at 24 or 28uC in vitro. At these two temperatures, all hybrid seedlings from both crosses died, whereas the parental species grew normally (Table 5, Figure 2). Lethal symptoms in hybrid seedlings of N. occidentalis R6N. sylvestris = grown at 24uC appeared approximately 18 DAG as browning of the hypocotyl ( Figure 2B). A few days later, chlorosis of leaves was observed. We identified this type of lethality as Type II. For hybrid seedlings of N. occidentalis R6N. tomentosiformis =, lethal symptoms at 24uC appeared as fading of shoot color at about 5 DAG and thus, the observed lethality was identified as Type V ( Figure 2E). Within each cross, the same characteristic symptoms appeared at 24 and 28uC (Table 5).

Discussion
Possible involvement of a dual lethal system causing hybrid lethality in crosses between N. occidentalis and N. tabacum Hybrid seedlings of N. occidentalis R6N. tabacum = and N. occidentalis R6N. tomentosiformis = exhibited hybrid lethality that was characterized by the fading of shoot color. As this characteristic was distinct from those of the four known types of lethality in Nicotiana [2], we designated this type of hybrid lethality as Type V. Notably, Type V lethality is also temperature sensitive, similar to Types I, II, and III. The type of lethality observed in the cross N. occidentalis R6N. tabacum = (Type V) was different from that (Type II) typically observed in crosses between species of section Suaveolentes and N.
tabacum [2,22]. In addition to lethality type, the cause of hybrid lethality in N. occidentalis R6N. tabacum = was also unique. As mentioned, we previously found that one or more genes on the Q chromosome in the S subgenome of N. tabacum were responsible for hybrid lethality in crosses of nine species in section Suaveolentes [20][21][22]. However, here, hybrid seedlings of N. tabacum R6N. occidentalis = were inviable, even when they lacked the Q chromosome. Surprisingly, N. occidentalis yielded inviable hybrids after crossing with both progenitors of N. tabacum. Characteristic symptoms of hybrid lethality in the cross N. occidentalis R6N. tabacum = were similar to those appearing in the cross N. occidentalis R6N. tomentosiformis =, strongly suggesting the involvement of gene(s) in the T subgenome. Nevertheless, we could not rule out the possibility that gene(s) in the S subgenome are also responsible for hybrid lethality in the cross N. occidentalis R6N. tabacum =. As lethal symptoms in the cross N. occidentalis R6N. tomentosiformis = appeared faster than in the cross N. occidentalis R6N. sylvestris =, the phenotype of Type V lethality might be preferentially expressed in the cross N. occidentalis R6N. tabacum =. If this supposition is true, the hybrid lethality observed in the cross between N. occidentalis and N. tabacum is the first reported for a dual lethal system in the same cross-combination. Furthermore, the causal chromosome in the S subgenome of N. tabacum may be the Q chromosome, because hybrid lethality in the cross N. occidentalis R6N. sylvestris = was Type II, which is often observed in crosses between species of section Suaveolentes and N. tabacum [2,22]. Although further evidence is needed, we conclude that genes from both the S and T subgenomes of N. tabacum are responsible for hybrid lethality in the cross between N. occidentalis and N. tabacum.
The viability and reproductive fitness of hybrids are often compromised when they are derived from parents that differ in ploidy levels [37]. Such dosage-sensitive incompatibility in interploidy crosses arises in the absence of any allelic diversity and is under genetic control [37,38]. Crosses between N. occidentalis and the two progenitors of N. tabacum were both interspecific and interploidal, because N. occidentalis is an allopolyploid, whereas N. sylvestris and N. tomentosiformis are diploids. Thus, it is difficult to determine whether hybrid lethality arose due to ploidy differences between the parental species. However, as we observed the identical hybrid lethality phenotypes in interploidy and same ploidy crosses, ploidy differences are not likely to have contributed to the observed hybrid lethality in crosses between N. occidentalis and the two progenitors of N. tabacum.

Unilateral incompatibility in crosses between N. tabacum and Suaveolentes species
Unilateral incompatibility or unilateral incongruity (UI) is often observed in interspecific crosses in Nicotiana. In crosses between N. tabacum and Australian species of section Suaveolentes, including N. occidentalis, pollen of Suaveolentes species is often compatible with the N. tabacum pistil, but the reciprocal cross is incompatible. UI in Nicotiana arises from: (1) self-incompatibility [39], (2) differences in pistil length and pollen tube growth rate [40,41], and (3) morphological abnormalities of pollen tubes, such as winding, swelling, and irregular callose deposition [42,43]. Self-incompatibility is unlikely to be a factor in UI for crosses between N. tabacum and Australian species of section Suaveolentes, because both N. tabacum and Suaveolentes species are self-compatible. In crosses using N. tabacum as the female parent, pollen from six short-pistil species, N. benthamiana, N. debneyi, N. goodspeedii, N. maritima, N. occidentalis, and N. velutina, did not produce seeds, while pollen from three long-pistil species, N. excelsior, N. gossei, and N. megalosiphon, occasionally produced seeds (pollen from N. tabacum is compatible with pistils of all of these short-and long-pistil species) [21,22,33]. Therefore, pistil length and pollen tube growth rate might be involved in UI, although this is unlikely to be the only factor, as we observed that many flowers of long-pistil species dropped after pollination with N. tabacum pollen. Although morphological abnormalities of pollen tubes are observed in other crosses within the genus Nicotiana, we have no data on pollen tubes for the crosses between N. tabacum and Suaveolentes species.

Evolutionary process of hybrid lethality in section Suaveolentes
Of the 12 Suaveolentes species used in the present and previous studies [20][21][22]33], only three species (N. occidentalis, N. benthamiana, Table 3. Relationship between the Q chromosome and hybrid lethality in crosses between N. tabacum and N. occidentalis. and N. fragrans) gave results different from those of the remaining 9 species. N. benthamiana and N. fragrans were the only species that produced viable hybrids after crosses with N. tabacum. This result is surprising, as section Suaveolentes presumably originated from a single polyploid event [26]. In a previous study, we concluded that the common gene(s) that trigger hybrid lethality by interaction with gene(s) on the Q chromosome should be widely distributed in species of section Suaveolentes [22]. Based on this conclusion, we suspect that the progenitor(s), namely N. sylvestris and/or section Trigonophyllae, which were involved in the formation of section Suaveolentes, might have possessed the gene(s) triggering hybrid lethality, whereas gene loss might have occurred during diploidization of the genome on polyploid establishment or during the subsequent divergence of N. benthamiana and N. fragrans [22]. However, it is also possible that the progenitors do not possess causal gene(s) for hybrid lethality in crosses with N. tabacum. Information on phylogeny is critical to estimate the evolutionary order and timing of causal genetic changes underlying reproductive isolation [44]. In three phylogenetic trees respectively constructed by analyses of sequence data for ITS regions, plastid genes, and ncpGS, section Suaveolentes formed a monophyletic group with the African species, N. africana, as a sister to the remaining species of section Suaveolentes [14,16,27]. More recently, Marks et al. [45] reported the results of phylogenetic analyses of section Suaveolentes based on the combined data set of morphological characters, chromosome number, and nodes that received strong bootstrap support in trees constructed from individual analyses of sequences of ITS regions, plastid genes, and ncpGS. In their tree, which was constructed using N. africana as the functional outgroup, the South Pacific species N. fragrans was the basal lineage in the Australian and South Pacific clade of section Suaveolentes. In addition, the Australian species N. debneyi (New Caledonia, Lord Howe Island, and Eastern Australia) was a sister species to all other endemic Australian species, including N. occidentalis and N. benthamiana.
Taking into account the results of recent phylogenetic analyses [45] and the crosses with N. tabacum performed here, we propose a new model for the evolutionary process leading to hybrid lethality in section Suaveolentes (Figure 3). It has been reported that the allotetraploid ancestor of the section, of which the maternal and paternal progenitors are N. sylvestris and section Trigonophyllae, respectively, occurred in South America [16,27]. In our proposed model, we considered that the allotetraploid ancestor did not cause hybrid lethality after crosses with N. tabacum. The appearance of Type II lethality in the lineages leading to N. africana and the Australian clade possibly occurred after the divergence of N. africana, N. fragrans, and the remaining species in section Suaveolentes ( Figure 3). However, we cannot deny the possibility that Type II lethality appeared in the allotetraploid descendent after the divergence of N. fragrans, and the allotetraploid descendent subsequently dispersed to Africa and Australia. Type II lethality may be caused by interaction with the Hla1-1 or other alleles found in the HLA1 locus, which was originally identified in N. debneyi [33], and gene(s) on the Q chromosome of N. tabacum. In Australia, an explosive radiation of taxa occurred that was largely accompanied by dysploid reductions, likely due to chromosomal fusions [27]. During the process, additional genetic changes that reinforced postzygotic isolation with N. tabacum accumulated in the lineage leading to N. occidentalis, leading to the appearance of Type V lethality (Figure 3). Conversely, loss of Type II lethality gave rise to the lineage leading to N. benthamiana.
Although the considerable amount of accumulated phylogenetic data for section Suaveolentes has allowed us to propose an evolutionary model of hybrid lethality, the present data are insufficient to definitively determine the origin and interspecies relationships of this section. The accumulation of additional phylogenetic data and a greater understanding of the mechanisms underlying hybrid lethality will allow further clarification of how hybrid lethality has evolved through speciation in section Suaveolentes.

Interspecific crosses
Conventional crossing and sowing were carried out as follows: flowers of plants used as maternal parents were emasculated one day before anthesis and pollinated with the pollen of paternal parent plants. F 1 seeds were soaked in a 0.5% gibberellic acid Table 5. Viability of hybrid seedlings generated from crosses between N. occidentalis and two progenitors of N. tabacum at different temperatures. Test-tube pollination in combination with ovule culture was performed as previously described [47] to obtain hybrid seedlings between N. tabacum monosomic plants and N. occidentalis. Anthers of N. occidentalis plants used as paternal parents were aseptically excised from still-closed flowers and stimulated to dehisce in an incubator held at 28uC. Flowers of monosomic plants used as maternal parents were emasculated one day before anthesis. On the next day, flowers of monosomic plants were collected and their corolla, sepals, and styles were removed. The ovaries were surface-sterilized with 70% ethanol for 30 s followed by a 5% sodium hypochlorite solution for 5 min. The ovary walls were peeled back to expose the placentas with intact ovules and the ovaries were then placed in Petri dishes containing 8 ml of medium supplemented with 3% sucrose and 0.8% agar (pH 5.8). Pollen of N. occidentalis was spread on the surface of the placentas, which were then maintained at 28uC under continuous illumination. Fertilized and enlarged ovules were excised from placentas 10 to 14 days after pollination and cultured in Petri dishes containing 8 ml of 1/2 MS medium supplemented with 3% sucrose and 0.8% agar (pH 5.8) at 28uC under continuous illumination.

Cultivation of hybrid seedlings
Hybrid seedlings and parental species were cultured at 24 or 28uC under continuous illumination. Several seedlings were transferred to flat-bottomed test tubes (25-mm diameter, 100-mm length) that contained 10 ml of 1/2 MS medium supplemented with 1% sucrose and 0.2% Gelrite (pH 5.8) immediately after germination and were cultured at 24, 28, or 34uC under continuous illumination. Seedlings cultured at 34uC for 30-60 DAG were then further cultured at 28uC under continuous illumination.

PCR analysis
Total DNA was extracted from the leaves of each plant using a cetyltrimethylammonium bromide-based method [48]. Four Qchromosome-specific STS markers, QCS1, QCS2, QCS3, and QCS4 [20,35], were detected by PCR as follows. Reaction mixtures consisted of 16 ThermoPol reaction buffer (New England Biolabs, Tokyo, Japan), 0.2 mM each dNTP, 0.2 mM each primer, 20 ng template DNA, and 0.5 U Taq DNA polymerase (New England Biolabs) in a total volume of 20 ml. PCR amplification was performed using a PC-818A Program Temp Control System (Astec, Fukuoka, Japan) programmed for 3 min at 94uC for initial denaturation, followed by 35 cycles of 30 s at 94uC, 30 s at 60uC, and 30-90 s at 72uC, with a final 5min extension at 72uC. PCR products were separated by electrophoresis in 1.5% agarose gels with TBE buffer and were then visualized by staining with ethidium bromide.

Ethics
The ethics committee of Osaka Prefecture University specifically waived the need for consent.  [45]. Gains of Type II lethality were established in the lineage leading to N. africana and the lineage leading to Australian clade. Subsequently, gain of Type V lethality was established in the lineage leading to N. occidentalis. In the lineage leading to N. benthamiana, loss of Type II lethality was established. doi:10.1371/journal.pone.0036204.g003