Nicotiana debneyi has a single dominant gene causing hybridlethality in crosses with N. tabacum

Takahiro Iizuka • Tsutomu Kuboyama •

Wataru Marubashi • Masayuki Oda •

Takahiro Tezuka

Received: 5 August 2011 / Accepted: 28 October 2011 / Published online: 4 November 2011

� Springer Science+Business Media B.V. 2011

Abstract To elucidate the genetic mechanism

of hybrid lethality observed in hybrids between

cultivated tobacco, Nicotiana tabacum, and wild

tobacco species in the section Suaveolentes, genetic

analyses were conducted through the triple cross of the

hybrids of wild species, including N. benthamiana and

N. fragrans, and N. tabacum. N. benthamiana and

N. fragrans produced only viable hybrids after cross-

ing with N. tabacum. Subsequently, N. benthamiana

and N. fragrans were crossed with N. africana,

N. debneyi, and/or N. suaveolens, which produced

inviable hybrids after crossing with N. tabacum.

Hybrids of the wild species were obtained from four

of the six cross combinations. Only when hybrid plants

of N. debneyi 9 N. fragrans, whose hybridity was

confirmed by morphological characteristics, random

amplified polymorphic DNA analysis, and chromo-

some observation, were crossed with N. tabacum,

triple hybrids were obtained and segregated 1:1

(lethal:viable). Based on these results, a single dom-

inant gene, designated Hybrid Lethality A1 (HLA1), in

N. debneyi was found to control hybrid lethality by the

interaction with gene(s) on the Q chromosome in

N. tabacum. This represents the first report to identify a

causal gene for hybrid lethality in the genus Nicotiana.

Keywords Genetic analysis � Hybrid lethality �Interspecific hybridization � Nicotiana section

Suaveolentes � Tobacco � Triple cross

Introduction

Hybrid lethality is a type of reproductive isolation

mechanism that has been observed in several plant

species, including Nicotiana species (Tezuka et al.

2010; Yamada et al. 1999), rice (Kuboyama et al. 2009),

wheat (Mizuno et al. 2010), cotton (Song et al. 2009),

and Arabidopsis thaliana (Bomblies et al. 2007). This

mechanism often causes the death of hybrid plants and

can be a barrier for the introduction of desirable genes

into cultivated species by wide hybridization.

In the genus Nicotiana, which is a commercially

important member of the family Solanaceae, many

attempts at interspecific hybridization have been

made, with several of the cross combinations resulting

in hybrids displaying abnormal growth and even death

(Tezuka et al. 2010; Yamada et al. 1999). Hybrid

lethality in Nicotiana species is classified into the

following four types based on surface symptoms: Type

T. Iizuka � M. Oda � T. Tezuka (&)

Graduate School of Life and Environmental Sciences,

Osaka Prefecture University, Sakai, Osaka 599-8531,

Japan

e-mail: tezuka@plant.osakafu-u.ac.jp

T. Kuboyama

College of Agriculture, Ibaraki University,

Ami, Ibaraki 300-0393, Japan

W. Marubashi

School of Agriculture, Meiji University,

Kawasaki, Kanagawa 214-8571, Japan

123

Euphytica (2012) 186:321–328

DOI 10.1007/s10681-011-0570-3

I, browning of the shoot apex and root tip; Type II,

browning of the hypocotyl and roots; Type III,

yellowing of true leaves; and Type IV, formation of

multiple shoots (Yamada et al. 1999).

In Nicotiana section Suaveolentes, there are at least

nine wild species, Nicotiana africana, N. debneyi,

N. excelsior, N. goodspeedii, N. gossei, N. maritima,

N. megalosiphon, N. suaveolens, and N. velutina,

which produce inviable hybrid seedlings exhibiting

Type II lethality after reciprocal crosses with culti-

vated tobacco, N. tabacum. This indicates that hybrid

lethality occurs due to interaction of the nuclear

genomes of N. tabacum and the wild species, and that

cytoplasmic factors are not involved (Tezuka and

Marubashi 2004, 2006a, b; Tezuka et al. 2010). When

these nine wild species were crossed with the

N. tabacum monosomic lines, Haplo-Q and/or F1

progeny derived from the cross Haplo-Q 9 N. taba-

cum ‘Samsun NN’, which only have one Q chromo-

some, two types of hybrid plants were obtained: viable

hybrid plants lacking Q chromosomes and inviable

hybrid plants possessing Q chromosomes. From these

results, the Q chromosome of N. tabacum appears to

encode a gene or genes causing hybrid lethality in

crosses between N. tabacum and the above nine wild

species of the section Suaveolentes (Tezuka and

Marubashi 2006a; Tezuka et al. 2007, 2010).

In contrast to the wild species described above, the

wild species N. fragrans, which also belongs to the

section Suaveolentes, does not produce inviable

hybrid seedlings on crossing with N. tabacum (Tezuka

et al. 2010). Thus, it is considered that N. fragrans

does not possess a factor causing hybrid lethality.

Additionally, DeVerna et al. (1987) reported that

N. benthamiana (section Suaveolentes) also yielded

viable hybrid plants after crossing with N. tabacum,

although it is unclear whether all of the obtained

hybrid plants were viable.

Genetic analysis for hybrid lethality has been

conducted in some plant species, including rice

(Ichitani et al. 2007; Kuboyama et al. 2009), wheat

(Mizuno et al. 2010), cotton (Song et al. 2009), and

A. thaliana (Bomblies et al. 2007). In these species,

hybrid lethality is caused by the interaction between

two dominant complementary genes from parental

plants. However, in Nicotiana species, the number of

genes responsible for hybrid lethality is not clear, as

hybrid seedlings expressing lethality typically die in

the cotyledonary or later stages, while the viable

hybrid plants incidentally obtained from crosses that

usually yield inviable hybrid seedlings were com-

pletely sterile (Tezuka and Marubashi 2006b).

As N. fragrans and N. benthamiana produce viable

hybrid plants after crossing with N. tabacum, these

two species are potentially useful for genetic analysis

of hybrid lethality in the genus Nicotiana. When

N. fragrans or N. benthamiana are crossed with wild

species producing inviable hybrid plants after crossing

with N. tabacum, it is expected that the gene(s) respon-

sible for hybrid lethality would be heterozygous in the

resulting F1 hybrids. Subsequent crossing between the

F1 hybrids and N. tabacum to form triple hybrid plants

would then result in the segregation of the gene(s),

allowing for their identification.

Here, we attempted to determine the identity of

genes in wild tobacco species that are responsible for

hybrid lethality in the crosses between N. tabacum and

several wild species in the section Suaveolentes. In

addition, we investigated whether N. benthamiana is

useful for the genetic analysis of hybrid lethality

by evaluating crosses between N. benthamiana and

N. tabacum.

Materials and methods

Plant materials

Five wild species of Nicotiana section Suaveolentes

were included in this study. Three species, N. africana

(2n = 46), N. debneyi (2n = 48) and N. suaveolens

(2n = 32), were known to possess causal gene(s)

for hybrid lethality, one (N. fragrans, 2n = 48)

was known not to possess the gene(s), and one

(N. benthamiana, 2n = 38) was suspected of not

possessing the gene(s). N. tabacum (2n = 48, SSTT)

‘Red Russian’ and ‘Samsun NN’ were used to test the

expression of hybrid lethality. As a preliminary part

of the study, N. benthamiana was crossed with

N. tabacum ‘Red Russian’ in both directions to

investigate whether the resulting hybrid seedlings

expressed lethality.

To obtain interspecific hybrids between wild spe-

cies, N. benthamiana was crossed with N. africana and

N. suaveolens as the male parents. N. fragrans was

also crossed with N. africana and N. debneyi in both

directions. The F1 hybrids obtained from these crosses

were then crossed with N. tabacum ‘Red Russian’ and

322 Euphytica (2012) 186:321–328

123

‘Samsun NN’ as the male parents to determine the

number of genes responsible for hybrid lethality

observed in the cross with N. tabacum.

Interspecific crosses

Flowers of plants used as female parents were

emasculated 1 day before anthesis and then pollinated

with the pollen of plants used as male parents. F1 seeds

obtained from crosses between N. benthamiana and

N. tabacum, and triple hybrid seeds were soaked in a

0.5% gibberellic acid (GA3) solution for 30 min and

then sterilized with 5% sodium hypochlorite for

15 min. The sterilized seeds were sown in petri dishes

(90 mm diameter) containing 25 ml half-strength MS

medium (Murashige and Skoog 1962) supplemented

with 1% sucrose and 0.2% Gelrite (pH 5.8), and were

cultured at 25�C under a photoperiod of 16 h light and

8 h dark (approx. 150 lmol m-2 s-1). Hybrid seeds

obtained from the crosses with wild species were sown

on a 1:1 (v/v) mixture of peat moss (Super Cell-Top V;

Sakata Seed Co., Kanagawa, Japan) and vermiculite

(Nittai Co., Osaka, Japan), and the plants were

cultivated in a greenhouse.

Chromosome analysis

To determine chromosome numbers, root tips were

pretreated with ion-exchange water for 24 h at 4�C,

followed by soaking in 2 mM 8-hydroxyquinoline for

4 h at 18�C, and were then fixed in ethanol/acetic acid

(3:1) overnight. The root tips were then hydrolyzed in

1 N HCl for 8 min at 60�C, stained with Schiff’s

reagent, and then squashed in 45% acetic acid. The

number of chromosomes in at least five root tip cells

for each plant was counted under a light microscope

(BX50; Olympus, Tokyo, Japan).

PCR analysis

Total DNA was extracted from the leaves of each plant

using the cetyltrimethylammonium bromide method

(Murray and Thompson 1980). The Q-chromosome-

specific DNA markers QCS1, QCS2, QCS3, and

QCS4 were detected as described by Tezuka and

Marubashi (2006a). Random amplified polymorphic

DNA (RAPD) analysis was carried out as described by

Williams et al. (1990) with some minor modifications

as follows. Briefly, 20 random 10-mer oligonucleotide

primers (Kit A) were obtained from Operon Technol-

ogies (Alameda, CA, USA). Reaction mixtures con-

tained 20 mM Tris–HCl (pH 8.8), 10 mM KCl, 2 mM

MgCl2, 10 mM (NH4)2SO4, 0.2 mM each dNTP,

0.5 lM primer, 20 ng template DNA, and 1.0 U

AmpliTaq DNA polymerase (Applied Biosystems,

Foster City, CA, USA) in a total volume of 20 ll. PCR

amplification was performed using a PC-818 thermal

cycler (Astec Corp., Fukuoka, Japan) programmed for

2 min at 94�C for initial denaturation, followed by 45

cycles of 30 s at 94�C, 30 s at 36�C, 2 min at 72�C,

and a final extension of 5 min at 72�C. PCR products

were separated by electrophoresis in a 1.5% agarose

gel in TBE buffer and stained with ethidium bromide

to visualize DNA bands. During analysis, only intense

and clear DNA bands were scored.

Phenotypic analysis

Segregation of lethal and viable plants obtained from

the cross between (N. debneyi 9 N. fragrans) 9 N.

tabacum were tested for goodness of fit to the expected

ratio at the 5% level using the v2 test. Hybrid seedlings

with and without browning of their hypocotyls and

roots were designated as ‘lethal’ and ‘‘viable’’,

respectively.

Results

Hybrid plants between N. benthamiana

and N. tabacum do not exhibit lethality

The results of reciprocal crosses carried out between

N. benthamiana and N. tabacum are shown in Table 1.

Although conventional crossing was successful using

N. tabacum as the male parent, flowers of N. tabacum

pollinated by N. benthamiana dropped approximately

7 days after pollination and no seeds were obtained,

suggesting that fertilization did not occur. The

obtained hybrid seeds germinated well, and all of the

hybrid seedlings were viable at 30 days after germi-

nation at 25�C (Table 1).

Twenty of the hybrid seedlings were randomly

selected and cultivated in a greenhouse. All of the

selected seedlings grew to maturity and came into

flower (Fig. 1a). The mature hybrid plants displayed

uniform morphological characteristics, with leaf and

flower shapes that were intermediate in appearance

Euphytica (2012) 186:321–328 323

123

between those of the parents (Fig. 1b–d). The chro-

mosomal analyses of five hybrid plants revealed that

each possessed 43 chromosomes, which is the sum of

the number of haploid chromosomes of the parents

(Fig. 1e).

Nicotiana benthamiana, N. tabacum, and the five

hybrid plants were also subjected to RAPD analysis

(Fig. 2). All 20 random primers gave RAPD patterns

showing distinct polymorphisms between the parents;

34 bands were detected only in ‘Red Russian’ and 40

bands were detected only in N. benthamiana. The five

hybrid plants had all 74 bands characteristic of both

parents, indicating that they were true hybrids. The

RAPD patterns obtained with primer OPA-14 are

shown in Fig. 2a.

The Q chromosome from N. tabacum causes hybrid

lethality in the crosses with most of the wild species in

the section Suaveolentes (Tezuka and Marubashi

2004, 2006a, b; Tezuka et al. 2010). To determine

whether hybrid seedlings of N. benthamiana 9

N. tabacum possessed the Q chromosome, isolated

DNA was analyzed for the presence of four Q

chromosome-specific DNA markers. The hybrids

had all of the marker bands that were detected in N.

tabacum, but none of those found in N. benthamiana

(Fig. 2b). Taken together, these results indicated

that N. benthamiana produced viable hybrids after

Table 1 Reciprocal

crosses between N. tabacum‘Red Russian’ and

N. benthamiana

Cross combination No. of

flowers

pollinated

No. of

capsules

obtained

No. of

hybrid

seeds sown

No. of hybrid

seedlings

Viable Lethal

N. benthamiana 9

‘Red Russian’

22 22 60 60 0

‘Red Russian’ 9

N. benthamiana22 0 – – –

Fig. 1 Hybrid from the cross of N. benthamiana 9 N. tabacum‘Red Russian’. a Shape of a hybrid plant that has grown to

maturity and flowered. Scale bar = 20 cm. b Leaves of

N. benthamiana (left), a hybrid plant (middle), and ‘Red Russian’

(right). Scale bar = 4 cm. c, d Flowers of N. benthamiana (left),a hybrid plant (middle), and ‘Red Russian’ (right). Scalebar = 1 cm. e Image of a root tip cell of a hybrid plant showing

the number of chromosomes. Scale bar = 100 lm

Fig. 2 Random amplified polymorphic DNA (RAPD) analysis

of the hybrids obtained from the cross between N. benthamianaand ‘Red Russian’. a Confirmation of hybrid formation by

RAPD analysis using primer OPA-14. b Detection of the

marker QCS1. M, DNA markers (k/Hind III and uX174/HaeIII). Lane 1, N. benthamiana; lanes 2–6, hybrid plants; lane 7,

‘Red Russian’

324 Euphytica (2012) 186:321–328

123

crossing with N. tabacum and would be a useful

species for the genetic analysis of hybrid lethality.

Interspecific crosses among wild species in section

Suaveolentes

Interspecific crosses were conducted between

N. benthamiana or N. fragrans and several wild

species in the section Suaveolentes that produce

inviable hybrid plants after crossing with N. tabacum.

As N. debneyi and N. fragrans have the identical

number of chromosomes and are closely related, and

because a high degree of chromosome pairing was

observed in the interspecific hybrids between N.

africana and N. fragrans (Gerstel et al. 1979), it was

expected that these cross combinations would produce

fertile hybrids. Additionally, a few other cross com-

binations were attempted (Table 2). Hybrid seeds

were obtained from four cross combinations,

N. benthamiana 9 N. africana, N. benthamiana 9

N. suaveolens, N. fragrans 9 N. africana, and

N. debneyi 9 N. fragrans, and were successfully

germinated to produce hybrid seedlings (Table 2).

Of the germinated hybrid seedlings from the four cross

combinations, 8, 1, 8 and 10, respectively, were

selected at random and cultivated in a greenhouse.

All of the hybrid plants came into flower.

N. debneyi has a single dominant gene causing

hybrid lethality in the cross between N. debneyi

and N. tabacum

The hybrid plants obtained from the four cross

combinations described above were crossed with

N. tabacum. Hybrid seeds were only obtained from

the interspecific crosses of (N. debneyi 9 N. frag-

rans) 9 N. tabacum, as none of the other crosses

yielded hybrid seeds (Table 3). All of the obtained

hybrid seeds germinated well.

The cross (N. debneyi 9 N. fragrans) 9 N. taba-

cum ‘Samsun NN’ yielded 163 hybrid plants, which

were segregated into 84 lethal and 79 viable plants,

while the cross (N. debneyi 9 N. fragrans) 9

N. tabacum ‘Red Russian’ gave 89 hybrid plants,

which were segregated into 46 lethal and 43 viable

plants (Tables 3 and 4). Both of the segregations fitted

a single dominant gene model, with a 1:1 (lethal:via-

ble) ratio at the 5% significance level. These results

indicated that N. debneyi has a single dominant gene

causing the hybrid lethality observed in the cross with

N. tabacum.

Analysis of hybrids obtained from the cross

between N. debneyi and N. fragrans

We next investigated the number of chromosomes and

the morphological characteristics of hybrid plants

resulting from the cross of N. debneyi 9 N. fragrans,

because these hybrids might be useful for the gener-

ation of an F2 population that is suitable to map the

hybrid lethality gene(s) of N. debneyi. The hybrid

plants had uniform morphological characteristics

(Fig. 3a) and displayed leaf and flower shapes that

were intermediate in appearance to those of the parents

(Fig. 3b–d). Five hybrid plants were analyzed for

chromosome number and it was revealed that all plants

possessed 48 chromosomes, which is the sum of the

number of haploid chromosomes of the parents (data

not shown).

RAPD analysis was also performed using a set of 20

random primers. The five hybrid plants gave RAPD

patterns showing distinct polymorphisms between

those of the parents; 34 bands were detected only

in N. debneyi and 36 bands were detected only in

Table 2 Interspecific

crosses among wild species

of section Suaveolentes

Cross combination No. of

flowers

pollinated

No. of

capsules

obtained

No. of

seeds

sown

No. of hybrid

seedlings

obtained

N. africana 9 N. fragrans 12 0 – –

N. fragrans 9 N. africana 7 5 442 24

N. benthamiana 9 N. africana 7 2 131 59

N. benthamiana 9 N. suaveolens 30 2 143 1

N. debneyi 9 N. fragrans 2 2 87 58

N. fragrans 9 N. debneyi 2 0 – –

Euphytica (2012) 186:321–328 325

123

N. fragrans. The five hybrid plants had all 70 bands

characteristics of both parents, indicating that they

were true hybrids. The RAPD patterns obtained with

primer OPA-16 are shown in Fig. 3e.

Fifteen flowers of N. debneyi 9 N. fragrans

hybrids were self-pollinated. Six capsules containing

F2 seeds with normal appearance were obtained,

indicating that the hybrids of N. debneyi 9 N. frag-

rans were both male and female fertile.

Discussion

Nicotiana debneyi produces inviable hybrid seedlings

after reciprocal crosses with N. tabacum, indicating

that an interaction between nuclear genomes causes

hybrid lethality and that cytoplasmic factors are not

involved (Tezuka and Marubashi 2006b). In the

present study, we determined that N. debneyi has a

single dominant gene responsible for the hybrid

lethality observed in these interspecific crosses. Tez-

uka et al. (2007) suggested that the Q chromosome of

N. tabacum has a gene or genes responsible for hybrid

lethality. Our findings are consistent with the conclu-

sion that the hybrid lethality observed in reciprocal

crosses between N. debneyi and N. tabacum is caused

by the interaction between a single dominant gene in

N. debneyi and gene(s) on the Q chromosome of

N. tabacum. We designated the causal gene of hybrid

lethality between N. tabacum and N. debneyi as HLA1

and assigned the N. debneyi allele that causes the

hybrid lethality as Hla1-1. We also tentatively desig-

nated the non-causal allele of N. fragrans and

N. tabacum as hla1-2, although further study is needed

to confirm whether this allele is shared between these

two species.

In the genus Nicotiana, other gene combinations

causing hybrid lethality also likely exist. In the cross

between N. repanda in the section Repandae and

N. tabacum, hybrid seedlings show Type III hybrid

lethality (Reed and Collins 1978; Yamada et al. 1999).

In addition, when N. repanda was crossed with two

ancestors of N. tabacum, N. sylvestris (2n = 24, SS)

and N. tomentosiformis (2n = 24, TT), inviable hybrid

seedlings were only obtained in the cross with

N. tomentosiformis, suggesting that the T subgenome

of N. tabacum is involved in hybrid lethality in the

cross between N. repanda and N. tabacum (Kobori and

Marubashi 2004). Therefore, genes or loci controlling

hybrid lethality in the cross between N. repanda and

N. tabacum would be different from those involved in

the cross between N. debneyi and N. tabacum, since

the Q chromosome is part of the S subgenome of

N. tabacum (Tezuka et al. 2007).

Gene(s) on the Q chromosome of N. tabacum also

cause hybrid lethality in the crosses with eight species,

N. africana, N. excelsior, N. goodspeedii, N. gossei,

N. maritima, N. megalosiphon, N. suaveolens, and

Table 3 Interspecific crosses between F1 hybrids of wild species of section Suaveolentes and N. tabacum

Cross combination No. of flowers

pollinated

No. of capsules

obtained

No. of seeds

sown

No. of seedlings

obtained

(N. debneyi 9 N. fragrans) 9 ‘Red Russian’ 4 2 120 89

(N. debneyi 9 N. fragrans) 9 ‘Samsun NN’ 8 4 200 163

(N. benthamiana 9 N. africana) 9 ‘Red Russian’ 22 0 – –

(N. benthamiana 9 N. africana) 9 ‘Samsun NN’ 40 0 – –

(N. benthamiana 9 N. suaveolens) 9 ‘Red Russian’ 22 0 – –

(N. fragrans 9 N. africana) 9 ‘Samsun NN’ 23 0 – –

Table 4 Phenotypic ratios for the two segregating populations from the cross of (N. debneyi 9 N. fragrans) 9 N. tabacum

Cross combination No. of hybrid seedlings obtained v2(1:1) P value

Viable Lethala

(N. debneyi 9 N. fragrans) 9 ‘Red Russian’ 43 46 0.1011 0.70-0.90

(N. debneyi 9 N. fragrans) 9 ‘Samsun NN’ 79 84 0.1534 0.50-0.70

a Number of hybrid seedlings with browning of their hypocotyls and roots

326 Euphytica (2012) 186:321–328

123

N. velutina, in the section Suaveolentes (Tezuka and

Marubashi 2006a; Tezuka et al. 2010). Section Suave-

olentes was revealed to be a monophyletic group based

on analyses of internal transcribed spacer (ITS)

regions, plastid genes, and nuclear-encoded chloro-

plast-expressed glutamine synthetase (ncpGS) (Chase

et al. 2003; Clarkson et al. 2004, 2010). Taken together,

these findings are consistent with the assumption that

these eight wild species in the section Suaveolentes

share the Hla1-1 allele identified in the present study

(Tezuka et al. 2010). In contrast, N. fragrans (Tezuka

et al. 2010) and N. benthamiana, which yield 100%

viable hybrid seedlings in crosses with N. tabacum,

possess the hla1-2 allele. Thus, N. fragrans and

N. benthamiana appear to be specific within the section

Suaveolentes with respect to hybrid lethality.

Reproductive isolation mechanisms restrict gene

flow and have important roles for speciation. A theory

for the development of these mechanisms, known as

the Bateson–Dobzhansky–Muller (BDM) model, pos-

its that genetic incompatibility in hybrids is caused by

deleterious interaction between two or more genes that

have evolved in different species sharing a common

ancestor (Coyne and Orr 2004). In some plant species,

including A. thaliana (Bomblies et al. 2007), cotton

(Song et al. 2009), lettuce (Jeuken et al. 2009), rice

(Kuboyama et al. 2009), and wheat (Mizuno et al.

2010), hybrid lethality is consistent with the BDM

model and is controlled by two complementary

dominant genes. Hybrid lethality observed here in

the cross between N. debneyi and N. tabacum is also

consistent with the BDM model, because it involves

the interaction between HLA1 and gene(s) on the Q

chromosome.

Hybrid sterility occurred in the hybrids among the

examined wild species of the section Suaveolentes,

excluding those resulting from the cross between

N. debneyi 9 N. fragrans. Unbalanced chromosome

sets are reported to cause hybrid sterility (Comai

2000). As only N. debneyi and N. fragrans have the

identical number of chromosomes (2n = 48) among

the wild species used in this study, and because these

two species are closely related, proper pairing of

chromosomes would have occurred in crosses of these

species, resulting in fertile hybrids.

Section Suaveolentes is monophyletic and consists

of closely related wild species (Chase et al. 2003;

Clarkson et al. 2004, 2010). Wheeler (1945) observed

a high degree of chromosome pairing in pollen mother

cells of interspecific hybrids among wild species

within the section. These results suggest that cross

combinations other than those attempted in the present

study may produce hybrids with a high degree of

chromosome pairing and fertility. We speculate that

N. benthamiana yields fertile F1 hybrids after crossing

with N. excelsior (2n = 38), as the morphological

characters, chromosome number, and ITS regions and

ncpGS gene of these two species closely related

(Ladiges et al. 2011). Thus, this potentially represents

another useful cross combination for the genetic

analysis of hybrid lethality.

Notably, F2 seeds were obtained from the cross

between N. debneyi and N. fragrans, suggesting that

the HLA1 locus can be mapped through linkage

analysis, if crossing over occurs. Further map-based

Fig. 3 Hybrid obtained from the cross of N. debneyi 9 N.fragrans. a Shape of the hybrid plant that has grown to maturity

and flowered. Scale bar = 15 cm. b Leaves of N. debneyi (left),a hybrid plant (middle), and N. fragrans (right). Scalebar = 4 cm. c, d Flowers of N. debneyi (left), a hybrid plant

(middle), and N. fragrans (right). Scale bar = 1 cm. e Confir-

mation of hybrid formation by RAPD analysis using primer

OPA-16. M, DNA markers (k/Hind III and uX174/Hae III).

Lane 1, N. debneyi; lanes 2–6, hybrid plants; lane 7, N. fragrans

Euphytica (2012) 186:321–328 327

123

cloning of HLA1 will aid in the understanding of the

mechanism of hybrid lethality observed in the genus

Nicotiana.

Acknowledgments We thank Japan Tobacco, Inc., Iwata,

Japan, for providing seeds of cultivated and wild species of the

genus Nicotiana. This work was partly supported by a Grant-in-

Aid for Young Scientists (Start-up; No. 20880024) from the

Japan Society for the Promotion of Science, Japan.

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