Biological Journal of the Linnean Society
, 2004,
82
, 599–606. With 1 figure
© 2004 The Linnean Society of London,
Biological Journal of the Linnean Society,
2004,
82
, 599–606
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Blackwell Science, LtdOxford, UKBIJBiological Journal of the Linnean Society0024-4066The Linnean
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599606
Original Article
GENOME EVOLUTION IN ALLOTETRAPLOID
NICOTIANA
K. Y. LIM
ET AL.
*Corresponding author. E-mail: a.r.leitch@qmul.ac.uk
Biological relevance of polyploidy: ecology to genomics
Edited by A. R. Leitch, D. E. Soltis, P. S. Soltis, I. J. Leitch and J. C. Pires
Genome evolution in allotetraploid
Nicotiana
KAR YOONG LIM
1
, ROMAN MATYASEK
2
, ALES KOVARIK
2
and ANDREW R. LEITCH
1
*
1
School of Biological Sciences, Queen Mary, University of London, London E1 4NS, UK
2
Institute of Biophysics, Academy of Sciences of the Czech Republic, CZ-61265 Brno, Czech Republic
Received 4 July 2003; accepted for publication 5 January 2004
The nuclear cytoplasmic interaction (NCI) hypothesis of genome evolution and speciation in plants states that newly
formed allopolyploids pass through a bottleneck of sterility and the fertile plants that emerge are fixed for species-
specific chromosome translocations. These translocations restore fertility and reduce negative effects of the maternal
cytoplasm on an alien paternal genome. Using fluorescent
in situ
hybridization and genomic
in situ
hybridization
and by reviewing published data, we test the NCI hypothesis using three natural
Nicotiana
allotetraploids (all
2
n
=
4
x
=
48,
N. arentsii
,
N. rustica
and several genotypes, including a feral plant and cultivars, of
N. tabacum
(tobacco)). We compare these data with three synthetic tobacco plants (Th37) that are F3 descendent progeny of an
allotetraploid formed from
Ǩ
N. sylvestris
(2
n
=
24)
¥
ǩ
N. tomentosiformis
(2
n
=
24). No intergenomic translocations
were observed in
N. arentsii
and
N. rustica
. An analysis of subtelomeric tandem repeats in these allotetraploids and
their putative parents shows minimal genetic changes; those that do occur may reflect evolution in the diploids or
the polyploids subsequent to allopolyploidy. All natural
N. tabacum
genotypes have intergenomic translocations.
This may reflect a large ‘genomic-shock’ generated by allopolyploidy involving widely diverged parental species. Two
of three synthetic tobacco plants had a translocation similar to that found in all cultivars of tobacco. This translo-
cation may be significant in tobacco fertility and may have been fixed early in tobacco’s evolution. But it is lacking
in the feral tobacco, which might indicate a polyphyletic origin or early divergence from all cultivars examined. Over-
all, only in tobacco is there any evidence that NCI may have influenced genome evolution, and here further data are
required to verify chromosome identity.
© 2004 The Linnean Society of London,
Biological Journal of the Linnean
Society
, 2004,
82
, 599–606.
ADDITIONAL KEYWORDS:
allopolyploid –
N. paniculata
–
N. undulata
–
N. wigandioides
– reticulate
evolution – satellite repeat.
INTRODUCTION
Gene conversion, chromosomal translocations and
sequence loss, gain, amplification and reduction have
all been associated with allopolyploidy and are per-
haps induced by allopolyploidy. But it remains unclear
whether changes are species-specific, ubiquitous,
accelerated in polyploids or simply reflect evolutionary
processes that occur in diploids and polyploids alike.
According to Gill’s (1991) nuclear cytoplasmic inter-
action (NCI) hypothesis, a newly formed polyploid
passes through a bottleneck of sterility, and the fertile
plants that emerge have ‘species-specific transloca-
tions’. These translocations, rather than ‘random
translocations’, are involved in restoring fertility and
cytoplasmic compatibility in the allopolyploid. In sup-
port of this argument, Jiang & Gill (1994a) identified
in two tetraploid wheats ‘species-specific’ transloca-
tions, and Leitch & Bennett (1997) noted that two cul-
tivars of
Nicotiana tabacum
(tobacco, cvs. 095-55 and
35466) analysed by Kenton
et al
. (1993) had several
translocations common to both.
Song
et al
. (1995) extended the NCI hypothesis in
studies of synthetic
Brassica
allopolyploids. In these
synthetic plants, restriction fragment length polymor-
phisms (RFLPs) indicated that the paternal genome
degenerates more rapidly than the maternal genome.
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600
K. Y. LIM
ET AL
.
© 2004 The Linnean Society of London,
Biological Journal of the Linnean Society,
2004,
82
, 599–606
However, generalizations are proving difficult to find.
Studies of synthetic hybrids and allopolyploids of
Triti-
cum
and
Aegilops
revealed fast, non-random and repro-
ducible elimination of chromosome- and genome-
specific sequences (Ozkan, Levy & Feldman, 2001;
Shaked
et al
., 2001; Levy & Feldman, 2004 – this
issue). This elimination was associated with the acti-
vation of retroelements and the loss of uncharacterized
RNA transcripts, the latter reflecting either gene loss
or epigenetic inactivation (Kashkush, Feldman & Levy,
2002, 2003). By contrast, amplified fragment length
polymorphism (AFLP) analyses of newly synthesized
Gossypium
allopolyploids (cotton) showed no evidence
of rapid genetic change associated with allopolyploidy
(Liu
et al
., 2001; Adams & Wendel, 2004 – this issue).
Is there evidence for species-specific translocations
that may have been selected in early
Nicotiana
allopolyploid evolution? The genus
Nicotiana
is an
ideal model to study patterns of evolution associated
with polyploidy because it has
c
. 70 diploid and poly-
ploid species (Goodspeed, 1954; Chase
et al
., 2003) and
provides clear examples of recurrent, including recent,
reticulate evolution (Chase
et al
., 2003). Here we anal-
yse three natural allopolyploids,
N. rustica
,
N. arentsii
and several cultivars of
N. tabacum
, including a feral
tobacco, and synthetic tobacco plants descended from
a plant made by Burk (1973). We use fluorescent
in
situ
hybridization (FISH) to localize tandem repetitive
sequences and genomic
in situ
hybridization (GISH) to
identify intergenomic translocations, including ‘spe-
cies-specific translocations’. GISH, which uses
labelled total genomic DNA from either one or both
putative parents of the allopolyploid as a probe, is a
powerful method to identify parental chromatin in the
allopolyploid. The method has been applied to a range
of natural and cultivated allopolyploids to determine
allopolyploid parentage and to identify the occurrence,
nature, and distribution of whole parental genomes,
chromosomes and introgressed chromosome segments
(Heslop-Harrison, 1992; Jiang & Gill, 1994b; Gill &
Friebe, 1998; Heslop-Harrison, 1998). Thus, GISH is
an ideal method to search for intergenomic species-
specific translocations and the influence of NCI on
early allopolyploid evolution. The work complements
that of Kovarik
et al
. (2004 – this issue), who exam-
ined ribosomal DNA (rDNA) evolution in the same
material.
RESULTS
N
ICOTIANA
TABACUM
Nicotiana tabacum
(tobacco) is an allotetraploid
(2
n
=
4
x
=
48) with two cytologically distinct parental
genomes, the S-genome and the T-genome (Kenton
et al
., 1993). Flower morphology, chromosome segrega-
tion patterns, GISH, chloroplast and mitochondrial
sequence data all suggest that a close relative of
N. sylvestris
(2
n
=
2
x
=
24, section
Alatae
) is the
maternal S-genome donor (Goodspeed, 1954; Bland,
Matzinger & Levins, 1985; Kenton
et al
., 1993;
Mosconne, Matzke & Matzke, 1996; Lim
et al
., 2000;
Chase
et al
., 2003). The paternal, T-genome donor of
tobacco is more controversial, with ancestors of
N. tomentosiformis
,
N. otophora
(both section
Tomen-
tosae
, 2
n
=
2
x
=
24) or an introgression hybrid impli-
cated (Kenton
et al
., 1993; Parokonny & Kenton,
1995).
Lim
et al
. (2000) conducted a detailed karyotype
analysis of all species in
Nicotiana
section
Tomentosae
using a suite of repetitive DNA probes and demon-
strated that the T-genome of tobacco was closely sim-
ilar to
N. tomentosiformis
and quite dissimilar from
N. otophora
. Murad
et al
. (2002) extended the analysis
to show that the T-genome was most like one accession
of
N. tomentosiformis
, carrying the tandem repetitive
sequences called NTRS, GRD5, GRD3 and GRD53.
Thus tobacco evolved from a particular genotype of
N. tomentosiformis
that existed after the common
ancestor of extant
N. tomentosiformis
.
Although
N. otophora
is not the T-genome donor of
N. tabacum
, there is a possibility that
N. otophora
DNA has introgressed into the T-genome. This would
explain Riechers & Timko’s (1999) data on putrescine
N
-methyltransferase gene families in tobacco, in
which one family member is found in tobacco and
N. tomentosiformis
and another in tobacco and
N. otophora.
Similarly, Ren & Timko (2001) observed
some matching AFLP bands in tobacco with
N. otophora
and others with
N. tomentosiformis.
How-
ever, introgression of
N. otophora
DNA into tobacco is
only one possible explanation of the data; another is
that there was independent loss of sequences in the
diploids after tobacco’s formation. Certainly if
N. otophora
is involved in tobacco’s ancestry, it has left
no cytological footprint detected to date. In addition,
there is no evidence from Tnt1 retrotransposon inser-
tion sites for introgressed N. otophora DNA (Melayah
et al., 2004 – this issue).
We analysed published and new GISH data to
search for species-specific translocations in tobacco
cultivars SR1 and Gatersleben (Mosconne et al.,
1996), 095-55, 35455, SR1 (Kenton et al., 1993), 095-
55 (Lim et al., 2000), a feral tobacco (Fig. 1A) from
Bolivia (kindly donated by Dr S. Knapp, Natural
History Museum, London), and three synthetic
tobacco plants (Fig. 1E). The synthetic tobacco plants
are descendant progeny (F3) of a single plant made
by crossing
Ǩ
N. sylvestris
(2n = 24) ¥
ǩ
N. tomentosiformis (2n = 24) and the F1 converted to a
fertile amphidiploid by in vitro callus culture (F0)
(Burk, 1973). A tally of intergenomic translocations in
all tobacco allopolyploids observed is shown in Table 1.
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GENOME EVOLUTION IN ALLOTETRAPLOID NICOTIANA
601
© 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 82, 599–606
T
able 1.
Karyotype details and c
hromosome translocations (inc
luding nomenc
lature synonyms) that occur in different lines of tobacco
Chromosomes
(per 1C genome)
0955-55
a/b
Samsun
NN
d
35466
a
SR1
a/c
Gatersleben
c
Bolivia
e
Synthetic
Th37-1
f
Synthetic
Th37-9
g
Synthetic
Th37-8
h
Overall karyotype
No
. of S-genome c
hromosomes
9/9
10
10
10/10
10
11 (10)
11
11
12
No
. of
T
-genome chromosomes
6/7
10
9
9/10
10
6
1
0
1
1
1
2
No
. of recombinant c
hromosomes
9/8
4
5
5/4
4
7
(8)
3
3
–
Recombinant c
hromosomes
S1/t;
ST3;
S1/ST3;
N
+
/+
––
–/–
–
–
–
–
–
S2/t;
S2/ST1;
ST1;
S2/t1
+
/++
+
+
/++
–
++
–
S11/t;
ST2;
S11/ST2;
S11/t2
+
/++
+
+
/++
+
–––
T1/s;
TS1;
T1/TS4;
N
+
/+
––
–/–
–
–
–
–
–
*T4/s
–/–
–
–
–/–
–
++
+
–
T5/s;
TS2;
T2/TS1;
N
+
/++
+
+
/–
–
–
–––
T6/s;
TS4;
T6/TS2;
T11/t4
+
/+
–
++
/++
+
?
–––
T7/s/s;
TS3;
T7/TS5;
N
+
/++
–
–/–
–
–
–
–
–
T9/s;
TS5;
T9/TS3;
T9/t3
+
/++
+
+
/++
+
+
+
–
N;
TS6;
T11/TS6;
N
+
/–
–
–
–/–
–
–
–
–
–
GISH data reveal the number of S-genome
,
T
-genome or recombinant c
hromosomes in different tobacco lines
, reported by
a
K
enton
et al
.
(1993
),
b
Lim
et al
.
(2000
),
c
Mosconne
et al
.
(1996
),
d
P
arokonny & K
enton (1995
),
or
e–h
shown here:
e
F
igure 1A;
f
synthetic tobacco (
F
ig
.
1E),
g
synthetic tobacco (
F
ig
.
1E),
Th37-9 is aneuploid,
2
n
=
49 with an extra
T
-genome chromosome;
h
synthetic tobacco
Th37-8 (not shown).
A breakdown of the recombinant c
hromosomes is given:
(
+
) presence or (–)
absence of individual recombinants
.
The nomenclature of these c
hromosomes is confused between publications
. Synonyms are given
in the order
Lim
et al
.
(2000
),
K
enton
et al
.
(1993
),
P
arokonny & K
enton (1995
),
Mosconne
et al
.
(1996
).
N
=
no name for c
hromosome given.
*T4/s is a translocation observed for the fi
rst time
here
.
The text of this manuscript uses the nomenc
lature of the fi
rst entry
.
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T4/s
T4/s
S2/t
T4/s
T9/s
TH37-1
Th37-9
A
B
E
D
C
F
I
H
G
N. undulata
NUNSSP
NUNSSP
NUNSSP
N. wigandoides
NUNSSP
U-genome
W-genome
GISH
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GENOME EVOLUTION IN ALLOTETRAPLOID NICOTIANA
603
© 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 82, 599–606
1. A similar chromosome, identified as T9/s, is found
in all N. tabacum allopolyploids analysed, includ-
ing the synthetic tobacco plants Th37-1 and Th37-
9. This may indicate an important translocation for
allopolyploid stability and have adaptive signifi-
cance. In each case the chromosome is identified by
the translocation. GISH reveals that the chromo-
some carries a T-genome centromere and is there-
fore, by definition, a T-genome chromosome. Its
identity can be further narrowed down by exclusion
of chromosomes with markers and by its small size.
Thus a T9 origin chromosome remains the best fit,
but the chromosomes carrying the translocation
between lines may not be homologous. Likewise,
there may be forced homology for chromosomes T6/
s and for S11/t in the natural tobacco plants (e.g.
S11/t, which carries an NOR, might be confused
with independent translocations on S10 or S12).
2. Chromosome T4/s is distinctive and carries a
marker generated by a tandem repeat in the long
arm that stains strongly by GISH. The chromosome
is found in the Bolivian tobacco (Fig. 1A) and in
synthetic plants Th37-1 and Th37-9 (Fig. 1E). This
translocation is not found in cultivated tobaccos
(Parokonny & Kenton, 1995; Mosconne et al., 1996;
Lim et al., 2004). Its independent evolution in
Bolivian tobacco and in the synthetic tobacco sug-
gests that the translocation may be significant, per-
haps representing a hotspot of recombination.
3. Chromosome S2/t is distinctive as it carries a large
T-genome translocation. This chromosome is found
in all cultivars of tobacco, but is absent in the feral
tobacco from Bolivia. Without independent mark-
ers it cannot be demonstrated unequivocally that
the chromosome is homologous in all cultivars, but
certainly it appears similar morphologically. To our
surprise, synthetic tobacco Th37-1 and Th37-9 car-
ries a similar translocation (Fig. 1E).
4. There are discrepancies in the numbers of translo-
cations reported for cv. 095-55 (Kenton et al., 1993;
Lim et al., 2000) and for cv. SR1 (Kenton et al.,
1993; Mosconne et al., 1996). In both instances the
discrepancies fall at easily identifiable chromo-
somes. The differences may have arisen through
cultivar divergence in the respective laboratories or
misidentification of the breeding lines.
5. There are fewer translocations in the synthetic
tobaccos compared with most cultivated varieties.
One synthetic tobacco plant (Th37.8, not shown)
had no detectable intergenomic translocations. The
Th37 population of synthetic tobacco remains het-
erozygous and is not yet fixed for any translocation.
Because of the lack of independent markers to con-
firm homology, no direct support can be given for the
NCI hypothesis and the occurrence of ‘species-specific’
translocations. Nevertheless, there does appear to be
surprising congruence in the data among tobaccos of
widely different origin, including those newly synthe-
sized. There is some evidence that GISH labelling of
the paternal T-genome is poor compared with the
labelling of the maternal S-genome in cultivated
tobacco (Parokonny & Kenton, 1995). This may sug-
gest degeneracy of the paternal genome, as inferred
from studies of synthetic Brassica allopolyploids (Song
et al., 1995). There was no reduction in the strength of
GISH labelling to the T-genome in the synthetic
tobacco (Fig. 1C).
N
ICOTIANA
RUSTICA
Goodspeed (1954) used morphology, karyotype analy-
ses and breeding experiments to propose that the
parents of N. rustica (2n = 4x = 48) were ancestors
of N. paniculata (2n = 2x = 24) and N. undulata
(2n = 2x = 24). Chloroplast gene sequencing and phy-
logenetic reconstructions of Nicotiana indicate that
Figure 1. Root tip metaphases after FISH using biotin-labelled probes (Cy3, red fluorescence) and digoxigenin-labelled
probes (FITC, green fluorescence). Blue fluorescence is the DAPI counterstain for DNA. A, GISH to N. tabacum collected
from Bolivia (N. sylvestris genomic DNA, green; N. tomentosiformis genomic DNA, orange). Note the numerous small S-
genome translocations to T-genome chromosomes (arrows, T4/s is identified and can be compared with the similar
chromosome in synthetic tobaccos in E) and T-genome translocations to S-genome chromosomes (arrow heads). B, GISH
to metaphase of N. rustica (N. paniculata genomic DNA, orange; N. undulata genomic DNA, green). Arrows show nucleolar
organizer regions that label with both probes (yellow). C, GISH to synthetic tobacco plant Th37-9 (N. tomentosiformis
genomic DNA, orange; N. sylvestris genomic DNA green), and D, DAPI image of the same metaphase. E, Chromosomes
carrying intergenomic translocations in plants TH37-1 and Th37-9. The intensely stained bands labelled by GISH (arrows)
provide a useful marker to identify the chromosomes (the orange/yellow band on the short arm of one homologue of Th37-
1 is rDNA and is discussed in detail in Skalická et al. (2003). F, NUNSSP labelling to N. arentsii; G, DAPI image and H,
after GISH in a reprobing experiment to identify the genomic origin of the chromosomes in the same metaphase
(N. undulata genomic DNA, green; N. wigandioides, red). I, Karyotypes of N. undulata (top), N. wigandioides (bottom).
The N. arentsii karyotype (rows 2–5), derived from the metaphase in F–H was split into U- and W-genomes (middle two
rows) based on the labelling patterns generated by GISH. The NUNSSP signal to these chromosomes is shown (rows
2–5). Scale bar = 10 mm.
᭣
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the maternal genome, the P-genome donor, may be an
ancestor of N. paniculata and/or N. knightiana (sec-
tion Paniculatae) (Aoki & Ito, 2000; Chase et al.,
2003). Phylogenies using internally transcribed
spacer sequences (ITS) of rDNA (Chase et al., 2003)
suggest that the paternal genome donor is an ancestor
of N. undulata and/or N. glutinosa, although Good-
speed (1954) placed N. glutinosa in an entirely differ-
ent section.
GISH to N. rustica metaphases, using total genomic
DNA probes from N. paniculata and N. undulata,
localized 24 P-genome chromosomes and 24 U-genome
chromosomes, respectively. There were no interge-
nomic translocations, nor any evidence for reduced
effectiveness of the probe labelling the paternal U-
genome compared with the labelling of the maternal
P-genome (Fig. 1B). We recently isolated three fami-
lies of repetitive sequences, two from N. paniculata
(NPAMBO and NPAMBE, both 180-bp monomer
repeats) and one from N. undulata (NUNSSP) (Lim
et al., 2004). FISH using these probes to N. paniculata
and N. undulata metaphases revealed that they
hybridized only to their species of origin and to subte-
lomeric locations on most chromosome arms (Lim
et al., 2004). The pattern of probe hybridization to
N. rustica was nearly the sum of that found in the two
parents. However, NPAMBO repeats occurred in a
lower copy number and in a slightly different distri-
bution to the P-genome of N. rustica compared with
N. paniculata.
N
ICOTIANA
ARENTSII
Based on flower and chromosome morphology, Good-
speed (1954) proposed that N. arentsii is a natural
allopolyploid (2n = 4x = 48) with ancestors related to
N. undulata
(2n = 2x = 24)
and
N. wigandioides
(2n = 2x = 24). Plastid gene sequencing and phyloge-
netic reconstructions of Nicotiana indicate that the
maternal genome donor of N. arentsii is closely related
to N. undulata (section Undulatae) (Aoki & Ito, 2000;
Chase et al., 2003). An analysis of the structure of the
intergenic sequences (IGSs) that occur between 45S
rDNA subunits reveals that IGSs in N. arentsii are
closely similar to N. undulata. Indeed all the IGSs of
N. wigandioides origin appear to have been lost by
sequence homogenization (Kovarik et al., 2004). To
provide further evidence in support of Goodspeed’s
(1954) hypothesis that N. arentsii is indeed an
allopolyploid derived from these parents, we used
GISH and total genomic DNA from each of the puta-
tive parents (Fig.
1H). To each metaphase of
N. arentsii, total genomic DNA from N. undulata
labelled 24 U-genome chromosomes and total genomic
DNA from N. wigandioides labelled 24 W-genome
chromosomes (Fig. 1I). There were no intergenomic
translocations, and no evidence that the paternal W-
genome is degenerate.
Nicotiana wigandioides,
N. undulata
and
N. arentsii are closely related species in phylogenetic
reconstructions of the genus (Chase et al., 2003). The
tandem repeat NUNSSP, isolated from N. undulata
(Lim et al., 2004), labels subtelomeric regions of chro-
mosomes of N. undulata
and
N. wigandioides,
although the distribution of signal differs between
species (Fig. 1I). By conducting multiple labelling
strategies, it is possible to identify the genomic origin
of the chromosomes and the distribution of NUNSSP
in N. arentsii (Fig. 1F–H). A comparison of the U-
genome with N. undulata and of the W-genome with
N. wigandioides (Fig. 1I) reveals little change in the
distribution pattern of NUNSSP.
DISCUSSION
Allopolyploidy combines the genomes of two species
into a common nucleus. Subsequent evolution can
potentially result in genome re-organization and chro-
mosomal rearrangement. Gill (1991) and Jiang & Gill
(1994a) observed common translocations in Triticum
allopolyploids and breeding lines, and postulated that
species-specific translocations arise to restore fertility
in newly formed allopolyploids. These arguments have
also been applied to N. tabacum to explain the occur-
rence of intergenomic translocations (Leitch & Ben-
nett, 1997; Lim et al., 2000) and the apparent
degeneracy of the T-genome, which labels less strongly
by GISH (Parokonny & Kenton, 1995; Leitch & Ben-
nett, 1997).
GISH data on a range of natural N. tabacum geno-
types reveal that all have intergenomic translocations.
The cultivars all have a large translocation to an S-
genome chromosome (S2/t), as do two of three
synthetic tobacco plants derived from the same popu-
lation of seeds (Table 1). The feral tobacco from Bolivia
is different in that it lacks this translocation, perhaps
indicating that tobacco is polyphyletic or that this
tobacco diverged before the cultivars examined. The
synthetic tobacco plants demonstrate that interge-
nomic translocations can occur early in allopolyploid
evolution. Whether any translocations are fixed as
‘species-specific translocations’ in tobacco will remain
unclear until further analyses and more direct evi-
dence for chromosome homology is found.
Intergenomic translocations may have arisen in
tobacco because its parents are widely diverged
[i.e. N. tomentosiformis section Tomentosae and
N. sylvestris section Sylvestres, cf. phylogenetic
schemes (Aoki & Ito, 2000; Chase et al., 2003;
Knapp, Chase & Clarkson, 2004)]. By contrast, the
parents of N. rustica and particularly N. arentsii,
with no intergenomic translocations, are closely
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GENOME EVOLUTION IN ALLOTETRAPLOID NICOTIANA
605
© 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 82, 599–606
related. Similarly, Tragopogon allotetraploids that
combine closely related diploid genomes do not dis-
play intergenomic translocations (Soltis et al., 2004 –
this issue). A hypothesis linking parental genome
divergence with intergenomic translocations runs
counter to the view that the more closely related the
parental genomes, the more likely there will be
translocations because of homoeologous chromosome
pairing and multivalent formation in meiosis. In
support of the hypothesis is that the union of
diverged genomes upon allopolyploidy can elicit
‘genomic-stress’, which in turn may activate trans-
posons and retrotransposons, DNA strand breaks
(McClintock, 1984; Melayah et al., 2004) and possi-
bly intergenomic translocations. It is conceivable
that the more similar the parental genomes, the
lower the ‘genomic-stress’. It may also be significant
that only in tobacco are the karyotypes of the
parental species substantially different, i.e.
N. tomentosiformis lacks terminal heterochromatic
repeats found in N. sylvestris and the karyotype is
bimodal. Perhaps this karyotype divergence gener-
ates ‘genomic-stress’ in the allopolyploid.
There are small differences in the distribution of
tandem repeats between the P-genomes of N. rustica
and the putative maternal species N. paniculata,
whereas the U-genome is nearly identical to that of
N. undulata (Lim et al., 2004). Similarly, there are
only minor differences between N. arentsii and its
putative parents N. undulata and N. wigandioides
(Fig. 1I). These probably reflect divergence between
the diploids and the allopolyploids. Perhaps accessions
or lines of parental diploids with even more similar
banding patterns remain to be found. The distribution
of tandem repeats in N. tabacum can be accounted for
by direct inheritance from the diploid parents and by
re-organizations arising from the intergenomic trans-
locations (as determined by GISH) (Lim et al., 2000;
Murad et al., 2002). Thus, changes in the distribution
and copy numbers of tandem repeats in the three Nic-
otiana allopolyploids compared with the putative
parental species are few. They are certainly minor
compared with the large-scale changes that occur over
time-scales of diploid Nicotiana speciation, as demon-
strated for Nicotiana section Tomentosae (Lim et al.,
2000). It is possible that molecular analyses such as
RFLP and AFLP would reveal further genetic change
associated with allopolyploidy, as has been demon-
strated for synthetic allopolyploids and hybrids of
Brassica (Song et al., 1995) and Triticeae (Ozkan
et al., 2001; Shaked et al., 2001).
ACKNOWLEDGEMENTS
We thank NERC and the Czech Academy of Sciences
(grants 204/01/0313 and 521/01/0037) for funding. We
thank Miss D. Saikia and Mr J. Clarkson for assis-
tance. We thank Professor V. Sissons (USDA) and Dr
Sandra Knapp (Natural History Museum, London) for
Nicotiana material.
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