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AFLP analysis of genetic polymorphism and
evolutionary relationships among cultivated and
wild Nicotiana species
Nan Ren and Michael P. Timko
Abstract: Amplified fragment length polymorphism (AFLP) analysis was used to determine the degree of intra- and
inter-specific genetic variation in the genus Nicotiana. Forty-six lines of cultivated tobacco (Nicotiana tabacum L.) and
seven wild Nicotiana species, including N. sylvestris, N. tomentosiformis, N. otophora, N. glutinosa, N. suaveolens,
N. rustica, and N. longiflora, were analyzed, using at least eight different oligonucleotide primer combinations capable
of detecting a minimum of 50 polymorphic bands per primer pair. The amount of genetic polymorphism present among
cultivated tobacco lines (N. tabacum) was limited, as evidenced by the high degree of similarity in the AFLP profiles
of cultivars collected worldwide. Six major clusters were found within cultivated tobacco that were primarily based
upon geographic origin and manufacturing quality traits. A greater amount of genetic polymorphism exists among wild
species of Nicotiana than among cultivated forms. Pairwise comparisons of the AFLP profiles of wild and cultivated
Nicotiana species show that polymorphic bands present in N. tabacum can be found in at least one of three proposed
wild progenitor species (i.e., N. sylvestris, N. tomentosiformis, and N. otophora). This observation provides additional
support for these species contributing to the origin of N. tabacum.
Key words: AFLP, evolution, genetic diversity, Nicotiana, tobacco, wild relatives of tobacco.
Résumé : L’analyse AFLP (polymorphisme de longueur des fragments amplifiés) a été employée afin de déterminer le
niveau de variation génétique intra- et interspécifique au sein du genre Nicotiana. Quarante-six lignées du tabac cultivé
(Nicotiana tabacum L.) et sept espèces sauvages du genre Nicotiana (N. sylvestris, N. tomentosiformis, N. otophora,
N. glutinosa, N. suaveolens et N. longiflora) ont été analysées à l’aide d’au moins huit combinaisons différentes
d’amorces oligonucléotidiques capables de détecter un minimum de 50 polymorphismes par paire d’amorces. La quan-
tité de polymorphisme génétique présent au sein du tabac cultivé (N. tabacum) était limitée vu la grande similitude
entre les profils AFLP des cultivars provenant de partout dans le monde. Six groupes majeurs ont été observés parmi
les tabacs cultivés et ces regroupements reposaient principalement sur l’origine géographique ainsi qu’en fonction de
certains caractères liés à la qualité manufacturière. Un plus grand polymorphisme génétique existe parmi les espèces
sauvages que parmi les formes cultivées. Des comparaisons deux à deux des profils AFLP des espèces de Nicotiana
sauvages et cultivées montrent que des bandes polymorphes présentes au sein du N. tabacum peuvent être trouvées
également chez au moins une des trois espèces sauvages ancestrales proposées (N. sylvestris, N. tomentosiformis et
N. otophora). Cette observation vient appuyer davantage l’hypothèse voulant que ces espèces aient été à l’origine du
N. tabacum.
Mots clés : AFLP, évolution, diversité génétique, Nicotiana, tabac, espèces sauvages apparentées au tabac.
[Traduit par la Rédaction] 571
Ren and Timko
Introduction
Tobacco (Nicotiana spp.) has been cultivated by man for
thousands of years and has served as a medicinal herb, trade
commodity, and crop plant in many different cultures. Within
the past several decades, this plant has found yet another
use, serving as a widely utilized model system in plant-cell
culture and genetic-engineering research. Because of its eco
-
nomic importance and value as a biological research tool,
numerous investigations have been undertaken to examine
its evolutionary origin and genome structure and organiza
-
tion.
The genus Nicotiana is a member of the family Solanaceae
and has been divided into three subgenera (Rustica, Tabaccum,
and Petunioides) containing over 64 recognized species
(Goodspeed 1954; Narayan 1987). Although tropical in ori
-
gin, tobacco can now be found growing from about 60ºN to
45ºS (Akehurst 1981). The most well-known species is
Nicotiana tabacum L., grown commercially in at least 97
countries around the world.
Nicotiana tabacum is a natural amphidiploid (2n = 48)
thought to have arisen by hybridization of wild progenitor
species (Gerstel 1960, 1963). One proposal, based upon anal
-
Genome 44: 559–571 (2001) © 2001 NRC Canada
559
DOI: 10.1139/gen-44-4-559
Received December 6, 2000. Accepted April 30, 2001.
Published on the NRC Research Press Web site at
http://genome.nrc.ca on July 11, 2001.
Corresponding Editor: F. Belzile.
N. Ren and M.P. Timko.
1
Department of Biology,
University of Virginia, Charlottesville, VA 22903, U.S.A.
1
Corresponding author (e-mail: mpt9g@virginia.edu).
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ysis of karyotypes, chromosome pairing, and segregation of
phenotypic characteristics in hybrids derived by crossing
various diploid species, suggests that Nicotiana sylvestris
(subgenus Petunioides, section Alatae) was the contributor
of the maternal genome (designated S in some literature),
with the parental genome (or T genome) coming from either
Nicotiana tomentosiformis or Nicotiana otophora (subgenus
Tabacum, section Tomentosae) (Goodspeed 1954; Gerstel
1960, 1963). Nicotiana tomentosiformis is thought to be the
more likely candidate species involved in the cross. Compara
-
tive biochemical studies, isozyme analysis, and examinations
of organellar (plastid and mitochondrial) genome organiza
-
tion support the viewpoint that N. sylvestris is the maternal
parent and N. tomentosiformis the paternal parent (Bland et
al. 1985; Okamuro and Goldberg 1985; Shinshi et al. 1988;
Sperisen et al. 1991). Analysis of molecular features, such as
repetitive DNA sequences and the structure of various nu
-
clear gene family members, has also been employed to trace
the molecular evolution of tobacco and study the genetic di
-
versity in genus Nicotiana (Kuhrová et al. 1991; Matassi et
al. 1991; Volkov et al. 1996, 1999a, 1999b; Komarnitsky et
al. 1998; Lim et al. 2000a, 2000b). In most cases, these
studies also concluded that N. tomentosiformis was the pro
-
genitor parental donor along with N. sylvestris.
A second possible origin for N. tabacum was suggested
by Kenton et al. (1993), based upon molecular cytogenetic
analysis of N. tabacum and its presumptive wild diploid pro-
genitor species using genomic in situ hybridization (GISH).
These authors found that total genomic DNA probes from
N. otophora gave stronger GISH signals when hybridized to
N. tabacum than those from N. tomentosiformis. Based on
this evidence Kenton et al. (1993) suggested that a portion of
the N. tabacum genome originated from N. otophora, and
proposed that the progenitors of N. tabacum may have been
N. sylvestris and an introgressed hybrid between
N. tomentosiformis and N. otophora. Similar conclusions
were reached by Jakowitsch et al. (1998). Analysis of the
structure and nucleotide sequences of the nuclear gene fam
-
ily encoding putrescine N-methyltransferase, a key enzyme
in nicotine formation, in N. tabacum and comparison with
gene family structure and sequence information from
N. sylvestris, N. tomentosiformis, and N. otophora support
the hypothesis that N. tabacum originated from a cross in
-
volving N. sylvestris and an introgressed hybrid between
N. tomentosiformis and N. otophora (Hashimoto et al. 1998;
Riechers and Timko 1999).
In contrast, studies employing fluorescence in situ hybrid
-
ization (FISH) by Lim et al. (2000a) found no evidence to
support the involvement of N. otophora and showed that the
T genome of N. tabacum was remarkably similar to
N. tomentosiformis and quite dissimilar from that of
N. otophora. Such a conclusion is also supported by recent
studies by Lim et al. (2000b) and Kitamura et al. (2000). In
the former study, the structure and intranuclear distribution
of 45S rDNA was analyzed in N. sylvestris and
N. tomentosiformis and compared with that present in
N. tabacum. This study concluded that N. sylvestris and
N. tomentosiformis alone are the progenitors of N. tabacum.
In the latter study, FISH analysis of 5S and 18S rDNA simi
-
larly indicated that the T genome of N. tabacum was derived
from N. tomentosiformis and not N. otophora.
With the development of tools for genome analysis at the
molecular level, such as random-amplified polymorphic DNAs
(RAPDs; Williams et al. 1990; Collins and Symons 1993;
Grando et al. 1996; Mengistu et al. 2000), amplified frag
-
ment length polymorphisms (AFLPs; Vos et al. 1995), sim
-
ple sequence repeat polymorphisms (SSRPs; Akkaya et al.
1992; Thomas and Scott 1993), and inverse sequence-tagged
repeat (ISTR) analysis (Sensi et al. 1996), it has become
possible to examine in greater detail the evolutionary origin
of plant genomes, as well as to access the degree of genetic
variability among related groups of plants. For example,
studies have been carried out in wheat (Barrett and Kidwell
1998; Kidwell and Fox 1998), Arabidopsis (Breyne et al.
1999; Erschadi et al. 2000), grape (Cervera et al. 1998), rice
(Zhu et al. 1998), cassava (Roa et al. 1997; Sanchez et al.
1999), sunflower (Hongtrakul et al. 1996), and numerous
other plants (Hill and Malmberg 1996; Powell et al. 1996;
Gagne et al. 1998; Xu et al. 2000), providing significant in
-
sight into phylogenetic relationships and the degree of ge
-
netic diversity among individuals and populations.
Numerous types of tobacco are grown commercially and
are defined to a large extent by region and (or) area of pro
-
duction, method of curing, and intended use in manufactur
-
ing, as well as by some distinct morphological characters
and chemical differences (Wernsman 1987). At the present
time, only limited information is available on the relation-
ship between morphological variability and genetic diversity
in genus Nicotiana. Furthermore, only a few attempts have
been made to examine the degree of relatedness between
cultivated and wild tobacco species at the molecular genetic
level. In the present work, AFLP analysis was used to deter-
mine the degree of genetic diversity that exists among 46
lines of cultivated tobacco (N. tabacum) and seven wild species
of Nicotiana representing the three subgenera of Nicotiana.
The findings show that greater genetic variation exists among
wild species of Nicotiana than among cultivated forms, and
that few genetic differences underlie the significant morpho
-
logical variability observed among cultivated forms of
tobacco. In addition, these studies provide further support
for an evolutionary origin of N. tabacum from a hybridiza
-
tion involving involving N. sylvestris, N. tomentosiformis,
and N. otophora.
Materials and methods
Plant material and DNA isolation
Seeds from 46 accession lines of cultivated tobacco representing
18 different countries from around the world were obtained from
Dr. Verne A. Sisson (U.S. Department of Agriculture Research
Laboratory, North Carolina State University, Raleigh, N.C.). A
summary of this material is presented in Table 1. Represented in
this collection are 12 different commercial types of tobacco, de
-
fined by their area and (or) country of production, intended use in
manufacturing (i.e., cigar filler, cigar wrapper), method of curing
(dark air-cured, fire-cured), and morphological and biochemical
characteristics (e.g., oriental, primative, etc.). In addition, we
obtained seeds of seven wild Nicotiana species, including
N. sylvestris, N. tomentosiformis, N. otophora, N. glutinosa,
N. suaveolens, N. rustica, and N. longiflora, which represent all
three subgenera of Nicotiana (Narayan 1987) (Table 2). Nicotiana
sylvestris, N. tomentosiformis, and N. otophora are likely progeni
-
tor species of N. tabacum (Riechers and Timko 1999).
© 2001 NRC Canada
560 Genome Vol. 44, 2001
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Tobacco plants used for DNA isolation were grown in a soil–
vermiculite mixture in the greenhouse under natural lighting condi
-
tions. Young leaves were collected from individual plants, frozen
in liquid nitrogen, and stored at –80°C until use in DNA preparation.
DNA was prepared from pulverized frozen leaf material (Riechers
and Timko 1999), and the purified DNA was resuspended in
10 mM Tris-HCl (pH 8.0) buffer containing 1 mM EDTA, for use
in the assays described below. The DNA concentration in samples
was determined spectrophotometrically by measuring absorbance
at 260 nm, using a Varian DMS200 spectrophotometer.
AFLP analysis
AFLP analysis (Zabeau and Vos 1993; Vos et al. 1995) was car
-
ried out using the AFLP Analysis System I (Life Technologies,
Inc), essentially as described in the manufacturer’s protocol. Pairwise
© 2001 NRC Canada
Ren and Timko 561
No. Accession code
a
Common name
Country of
origin Type
b
1 Xanthi U.S.A.
2 SD PI 552380 NC 95 U.S.A. Flue-cured
3 SD PI 552305 Cash U.S.A. Flue-cured
4 SD PI 543792 TN 90 U.S.A. Burley type
5 SD PI 552326 Burley 21 U.S.A. Burley type
6 SD PI 552360 Havana 142 U.S.A. Cigar binder
7 SD PI 552406 Swarr-Hibshman U.S.A. Cigar filler
8 SD PI 552619 Connecticut Broad U.S.A. Cigar wrapper
9 SD PI 552438 Madole U.S.A. Dark air-cured
10 SD PI 552377 Lizard Tail Orinoco U.S.A. Fire-cured
11 SD PI 551332 Catterton U.S.A. Light air-cured
12 SD PI 405003 Awa Japan Flue-cured
13 SD PI 405022 Hatano Japan Cigar filler
14 SD PI 511861 Okinawa Japan Primitive
15 SD PI 418596 Ma-Song-Ta China Flue-cured
16 SD PI 405676 Chatham India Flue-cured
17 SD PI 405636 Natu Cheroot India Primitive
18 SD PI 370281 Kutsaga 51 Zimbabwe Flue-cured
19 SD PI 404944 Correntino Argentina Cigar filler
20 SD PI 117568 Lampazo Argentina Other
21 SD PI 117661 Azul Brazil Flue-cured
22 SD PI 404964 Amerellinho Brazil Other
23 SD PI 405538 Gumo Brazil Flue-cured
24 SD PI 112132 Mariense Brazil Burley type
25 SD PI 405516 Ambalema Columbia Cigar filler
26 SD PI 113526 Cubano Columbia Flue-cured
27 SD PI 114356 Garcia Columbia Cigar filler
28 SD PI 404943 Barbasco Ecuador Cigar filler
29 SD PI 494148 Cordoba Mexico Cigar filler
30 SD PI 494148 Colorado Mexico Primitive
31 SD PI 481860 Orinoco Mexico Flue-cured
32 SD PI 494164 Tabacco Corriente Venezuala Cigar filler
33 SD PI 494150 Sirogo Australia Flue-cured
34 SD PI 405621 Criollo Cuba Cigar filler
35 SD PI 408943 Big Cuban Cuba Cigar wrapper
36 SD PI 225977 Largo Costa Rica Cigar wrapper
37 SD PI 355074 Itzepeque Costa Rica Cigar wrapper
38 SD PI 114013 Palmira Costa Rica Other
39 SD PI 114086 Smyma Greece Oriental
40 SD PI 114015 Perustza Gigante Italy Semi-oriental
41 SD PI 494148 Basma Turkey Oriental
42 SD PI 494150 Bursa Turkey Oriental
43 SD PI 494156 Izmir Turkey Oriental
44 SD PI 481860 Samsun Turkey Oriental
45 SD PI 405016 Trabzon Turkey Oriental
46 SD PI 112238 Prilep Yugoslavia Semi-oriental
a
From the U.S.Department of Agriculture, Agricultural Research Service, National Resources
Program.
b
The major manufacturing quality trait.
Table 1. Nicotiana tabacum accession lines investigated in this study.
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combinations of [
γ
-
32
P]ATP-labeled (New England Nuclear Life
Sciences Products, Boston, Mass.) EcoRI and MseI selective prim
-
ers with two, three, and four nucleotide additions after the cleavage
site were tested in our analysis. In our assays, the ligation reaction
was diluted 1:5 with buffer prior to initiating the preamplification
reaction; following the preamplification reaction, the reaction mix
-
ture was diluted 1:50 prior to selective amplification. The selective
amplification products were mixed with an equal volume of dye
reagent (98% (v/v) formamide, 10 mM EDTA, 0.025% (w/v)
bromophenol blue, and 0.025% (w/v) xylene cyanol), and aliquots
were separated by electrophoresis through 6% (w/v) polyacrylamide
gels containing 5 M urea and 1× TBE buffer (Sambrook et al.
1989). After electrophoresis, the gels were transferred to Whatman
3MM paper, dried under vacuum, and subjected to autoradiography
at –80°C.
Data analysis
AFLP profiles generated using the various primer combinations
were scored in terms of presence or absence of the polymorphic
band in each of the 46 accession lines and the wild Nicotiana spe-
cies. Genetic distance (D) was calculated using the software pro-
gram PAUP 4.0b (Swofford 1998), applying pairwise comparison
as described by Nei and Li (1979), using the formula: D
xy
=1–
[2N
xy
/(N
x
+ N
y
)], where N
xy
is the number of fragments (bands)
shared between individuals x and y, N
x
is the number of fragments
in x, and N
y
is the number of fragments in y. Cluster analysis and
construction of dendrograms showing phenetic relationships be
-
tween individuals (species) were performed using UPGMA (un
-
weighted pair group method using arithmetic averages; Sneath and
Sokal1973) and neighbor-joining methods of analysis. The reliabil
-
ity of the clusters was checked by bootstrap analysis using the soft
-
ware program PAUP 4.0b. Principal component analysis was
performed using the PRINCOMP procedures available in SAS ver
-
sion 8 (SAS Institute Inc. 1999).
Results
Levels of polymorphism detected in cultivated tobacco
Forty-six accession lines of cultivated tobacco (N. tabacum
L.), previously classified by their region and (or) area of pro
-
duction, method of curing, and intended use in cigar, ciga
-
rette, and related manufacturing industries (Table 1), were
examined by AFLP analysis using various pairwise combi
-
nations of
32
P-labeled EcoRI (E) and MseI (M) selective
primers with two (+2), three (+3), or four (+4) nucleotide
additions after the cleavage site. Primer combinations of E +
2andM+3,such as E-TG + M-CAG and E-TG + M-CTG,
generated profiles in which the amplification products were
overlapping or too dense to allow reliable scoring and there
-
fore not useful in our analysis. In contrast, combinations in
-
volvingE+3andM+4generated too few amplification
products (e.g., selective primer combinations E-AAC + M-
CAGC, E-AAC + CATG, and E-AAC + M-CTAG produced
25, 44, and 38 bands, respectively), of which only a small
proportion were polymorphic. Therefore, these combinations
did not provide sufficient coverage of the whole genome to
be informative.
Of the primer combinations tested, the most productive
and reproducible combinations that would be useful in the
analysis of the tobacco genome were determined to be E-
AGC + M-CAG, E-AGC + M-CTT, E-AGC + M-CGG, E-
AAC + M-CGT, and E-ACC + M-CAC. Using these prim
-
ers, a total of 460 AFLP bands were produced of which 119
(25.87%) were polymorphic. The size of the AFLP frag
-
ments generated ranged from approximately 50 to 700 bp
(Fig. 1). Compared with measured levels of polymorphism
among varieties–culitvars in other plant species (Roa et al.
1997; Cervera et al. 1998; Law et al. 1998; Breyne et al.
1999), the degree of polymorphism recognized among the
various accession lines of cultivated tobacco is relatively
low, suggesting that the large differences observed in so-
called “manufacturing quality traits” may result from a rela
-
tively small number of genetic differences among cultivars.
Data from analysis of the AFLP profiles generated using
five different primer combinations were used to calculate a
similarity matrix among the various accession lines. Pairwise
comparisons are shown for 22 of these in Table 3 and the
remainder can be found at http://www.people.virginia.edu/
~mpt9g/table3exp.xls. Using these same data, a dendrogram
showing the phenetic relationships among the 46 accessions
was created using PAUP 4.0b, and the reliability of the ag-
gregating method checked by bootstrap analysis (Fig. 2).
Based on these analyses, it appears that the collection of ac-
cessions analyzed contains unique but related individuals
that fall into one of six main clusters. The six clusters sepa-
rate largely on the basis of geographic distribution (pre
-
sumed geographic origin and (or) region of growth) and
morphology. Group 1 consists of two accessions, the
cultivars Xanthi (U.S.A.) and Awa (Japan). The relatedness
of these cultivars has not been previously noted. Group 2
contains six cultivars, NC95, Cash, Catterton, Havana 142,
Connecticut Broad, and Madole, all grown in the U.S. In
-
cluded among these are two flue-cured types (NC95 and
Cash), one dark air-cured type (Madole), one filler type
(Catterton), one cigar-binder type (Havana 42), and one
cigar-wrapper type (Connecticut Broad). Group 3 and group
6 are small groups containing some interesting pairings. For
example, in group 6, all three accession are cigar-types from
Central America, whereas group 3 consists of two Japanese
cultivars, one cultivar from Turkey that is an oriental type,
and one U.S. cultivar.
The two largest clusters are groups 4 and 5. Of the two
clusters, group 5 consists of cultivars loosely bound by geo
-
graphic origin and exhibiting a diverse set of manufacturing
quality traits. Eleven of the 19 accessions comprising group
5 originate in Central and South America, whereas six are
oriental or semi-oriental types from Mediterranean regions
(Italy, Turkey, Greece, and Yugoslavia). Group 4 is the most
variable and consists of several small subgroups. One sub
-
group is composed of two Burley filler types: Burley 21 and
Mariense. Another is composed of plants mainly of Asian
© 2001 NRC Canada
562 Genome Vol. 44, 2001
No. Accession code
a
Species Subgenus
1 PI 555569 N. sylvestris Petunioides
2 PI 555531 N. longiflora Petunioides
3 PI 230961 N. suaveolens Petunioides
4 PI 499162 N. rustica Rustica
5 PI 555572 N. tomentosiformis Tabacum
6 PI 302477 N. otophora Tabacum
7 PI 555505 N. glutinosa Tabacum
a
From the U.S.Department of Agriculture, Agricultural Research
Service, National Resources Program.
Table 2. Summary of the wild Nicotiana spp. investigated in this
study.
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origin and includes the flue-cured types Ma-Song-Ta (China),
Chathan (India), and Lizard Tail Orinoco (U.S.A.) and the
primitive type Natu Cheroot (India). Yet another subgroup
consists of plants of diverse geographic origins and charac
-
teristics (e.g., Sirogo from Australia, Swarr-Hibshman from
the U.S., Bursa from Turkey, etc.).
When another aggregating method (i.e., the neighbor-joining
method) was used for cluster analysis, only subtle differ
-
ences in tree structure were found (data not shown). Group
1, consisting of the cultivars Xanthi and Awa, still formed a
basal node. Group 2, containing the majority of the cultivars
of U.S. origin (NC95, Cash, Catterton, Havana 142, Con
-
necticut Broad, and Madole), also reemerged as a dominant
node, and the majority of cultivars forming group 4 remained
together. Groups 3, 5, and 6 were compressed into one larger
cluster, with each of the groups still recognizable as small
groupings within the larger cluster. Regardless of the method
used for cluster analysis, the three wild species remained
well separated from the various N. tabacum cultivars.
At the present time, the underlying genetic basis for most
manufacturing quality traits is not well defined. To deter
-
mine whether some primer combinations were more effec
-
tive in separating out genotypes with similar manufacturing
quality traits, principal component analysis (PCA) was car
-
ried out treating each primer combination pair as a unique
data set. As shown in Fig. 3, when the selective primer com
-
bination E-ACG + M-CTT was used, two clusters composed
of accessions designated as oriental and flue-cured were re
-
solved, with the oriental types mainly separated along princi
-
pal component 2 (PC2) and the flue-cured types were
resolved along PC1. In this analysis, PC1 and PC2 accounted
for 39% of the variation. Primer combination E-AAC + M-
CGT similarly resolved clusters composed of oriental and
flue-cured cultivars but, in this case, the major separation
was along PC2, with the first two principal components ac
-
counting for 30% of the variation. None of the other primer
combinations analyzed showed a similar ability to resolve
the accessions into clusters based on manufacturing quality
traits. Although a direct correlation would need to be dem
-
onstrated by further segregation analysis, our observations
suggest that these particular primer combinations likely tar
-
get those portions of the genome associated with characters
responsible for defining the oriental and flue-cured pheno
-
types. It is worth noting that the clustering of genotypes
© 2001 NRC Canada
Ren and Timko 563
Fig. 1. AFLP profiles showing the genetic polymorphism detected among cultivated accession lines of Nicotiana tabacum using the se
-
lective primer combination E-AGC + M-CAG. In the figure, the accessions are grouped by geographic origin, and the numerical desig
-
nation above each lane corresponds to the accession line listed in Table 1. The arrows indicate highly polymorphic bands within the
profiles.
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observed in the PCA plots based on the two primer combina-
tions E-ACG + M-CTT and E-AAC + M-CGT was consis-
tent with the groupings observed with both the UPGMA and
neighbor-joining methods using the complete data set.
Genetic polymorphism in wild species of Nicotiana
Table 2 lists the seven species of Nicotiana used in this
study. These seven species represent all three subgenera of
Nicotiana (Narayan 1987), and N. sylvestris, N. otophora,
and N. tomentosiformis are thought to be the likely progeni
-
tors of N. tabacum (Riechers and Timko 1999). Using the
same EcoRI and MseI primer combinations found effective
for analyzing the various N. tabacum accession lines, AFLP
profiles were generated for each of the wild species and their
patterns compared with those generated for the various to
-
bacco accession lines (Fig. 4). A total of 809 different AFLP
bands were recognized among the seven species, with more
than 95% being polymorphic between at least two species.
Based on the presence or absence of the amplified fragments
in the profiles of the various species, the genetic distances
among species were calculated (Table 4) and a dendrogram
constructed (Fig. 5). The seven wild species of Nicotiana
fall into two main clusters based on their genetic distance (D
value). One cluster consists of N. sylvestris, N. longiflora,
and N. suaveolens, all three of which have been classified
previously as belonging to subgenus Petunioides. The sec
-
ond cluster consists of N. otophora, N. tomentosiformis, and
N. glutinosa, which have been placed together previously in
subgenus Tabacum. The placement of N. rustica is less
certain. Taxonomic classifications based on comparisons of
morphological and biochemical phenotypic markers placed
this species in subgenus Rustica. However, based on its D
value, N. rustica shows greater affinity to members of sub-
genus Tabacum than to members of subgenus Petunioides,
but is not sufficiently different from either to merit its own
grouping.
It was also of interest that, with certain primer combina
-
tions, it was possible to recognize AFLP markers that were
conserved in all Nicotiana spp. and accessions tested, or that
were present only in one or a subset of subgenera. For exam
-
ple, the primer combination E-AGC + M-CAG yielded four
different fragments present in the profiles of all N. tabacum
accessions and wild Nicotiana species investigated in our
study (Fig. 4). In contrast, the selective primer combination
E-AGC + M-CAG yielded AFLP markers present only in
species classified as members of subgenera Tabacum or
Petunioides (Fig. 5).
Evolutionary origin of cultivated tobacco
The AFLP profiles of 22 different N. tabacum accession
lines and those of the presumed progenitor species of cultivated
tobacco (N. sylvestris, N. otophora, and N. tomentosiformis)
generated using the selective primer combination E-ACC +
M-CAC are shown in Fig. 6. Of the 82 different amplifica
-
tion products present in the AFLP profiles of the N. tabacum
lines, 21.9% (18/82) were polymorphic. The Venezuelan ac
-
cession Tobacco Corriente contained all 82 AFLPs and,
therefore, was chosen as the reference. A total of 97 amplifi
-
cation products were identified among the combined profiles
of N. sylvestris, N. otophora, and N. tomentosiformis.Ofthe
total of 82 fragments present in the Tobacco Corriente pro
-
file, only four were not represented in the profiles of at least
© 2001 NRC Canada
564 Genome Vol. 44, 2001
No. Accession or species 1 2 3 4 5 6 7 8 9 10
1 Cash 0
2 Burley 21 0.703* 0
3 Havana 142 0.473 0.968 0
4 Catterton 0.359 0.583 0.503 0
5 Awa 0.105 0.748 0.12 0.92 0
6 Okinawa 0.113 0.948 1.476 1.268 1.257 0
7 Ma-Song-Ta 0.645 0.499 0.946 0.636 0.841 0.85 0
8 Natu Cheroot 0.7 0.443 0.964 0.691 0.821 0.944 0.423 0
9 Kutsaga 51 0.672 0.64 1.007 0.737 0.942 0.688 0.509 0.49 0
10 Correntino 0.727 0.621 1.025 0.791 1.113 0.706 0.453 0.471 0.266 0
11 Amerellinho 0.73 0.699 1.029 0.794 1.079 0.709 0.529 0.621 0.267 0.321
12 Ambalema 0.723 0.73 1.104 0.864 1 1.045 0.444 0.688 0.735 0.753
13 Barbasco 0.823 0.642 1.123 0.886 1.097 0.727 0.584 0.714 0.357 0.375
14 Colorado 0.782 0.714 1.118 0.845 1.131 0.724 0.618 0.711 0.355 0.301
15 Tabacco Corriente 0.764 0.621 0.987 0.754 0.96 0.967 0.416 0.471 0.446 0.391
16 Sirogo 0.685 0.615 1.027 0.751 0.883 0.892 0.556 0.537 0.548 0.64
17 Big Cuban 0.846 0.589 1.072 0.835 0.968 0.976 0.568 0.512 0.56 0.578
18 Itzepeque 0.912 0.807 1.174 0.974 1.072 0.927 0.823 0.766 0.481 0.571
19 Smyma 0.767 0.661 0.991 0.831 1.156 0.783 0.64 0.696 0.448 0.429
20 Perustza 0.918 0.812 1.297 1.055 1.273 0.635 0.715 0.733 0.521 0.539
21 Bursa 0.758 0.575 1.061 0.748 0.841 0.812 0.479 0.535 0.546 0.49
22 Prilep 0.866 0.797 1.169 0.929 1.104 0.732 0.513 0.643 0.396 0.45
23 N. sylvestris 5.147 5.037 5.244 5.059 4.759 4.603 5.1 4.953 4.672 4.693
24 N. otophora 8.767 9.04 9.109 8.954 9.04 8.832 8.657 8.902 8.963 8.984
25 N. tomentosiformis 6.838 6.859 6.633 6.655 6.859 7.337 6.673 6.694 6.715 6.435
Note: Numbers in the first row of the table are equivalent to those in the first column indicating accession or species names.
Table 3. Genetic distance among accessions of cultivated tobacco and selected wild species of Nicotiana based on AFLP-profile analysis.
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one of the wild species. Similarly, only 19 of a total of 97
fragments found in the profiles of N. sylvestris, N. otophora,
and N. tomentosiformis could not be found in the Tobacco
Corriente profile. In pairwise comparisons, 65.85% (54/82)
of the bands present in N. sylvestris, 52.44% (43/82) of the
bands present in N. tomentosiformis, and 43.68% (35/82) of
the bands present in N. otophora were represented in the To
-
bacco Corriente profile. These data indicate that all three
progenitor species contributed information to the evolution
of the N. tabacum genome, with the greatest contribution be
-
ing made by N. sylvestris, the presumed maternal parent.
Similar results were found with other combinations of selective
primers and, as indicated in Table 3, estimates of genetic
distances (D values) based on the analysis of five different
primer combinations indicated that N. sylvestris is more closely
related to N. tabacum (D value of 0.0436–0.0530) than are
N. otophora (D value of 1.669–0.0865) or N. tomentosiformis
(D value of 1.160–0.0643). As noted above, N. otophora and
N. tomentosiformis clustered together, with N. sylvestris sep
-
arated from them but closer to N. tabacum.
Discussion
Although evolutionary relationships in the genus Nicotiana
have been analyzed previously and a significant amount of
morphological and cytological information is available, little
information is available on the extent of genetic variation
within commercially cultivated tobacco and between culti
-
vated forms of N. tabacum and its wild relatives. In this
study, we used AFLP analysis to gain insight into the degree
of intra- and inter-specific variation in this genus, as well as
to re-examine evolutionary relationships between some of its
members. Our studies show that, with appropriately defined
reaction conditions and specific combinations of selective
primers, it is possible to yield a sufficient number of poly-
morphic bands to allow meaningful comparison among culti-
vated tobacco accession lines and among species. In some
cases, a single primer combination was found to be capable
of providing a sufficient number of data points (>50 polymor
-
phic fragments) to distinguish between highly related indi
-
viduals. Using multiple primer combinations, we were able
to build data sets providing clear and conclusive relation
-
ships among accessions and defining evolutionary relation
-
ships among species.
It was suggested earlier that, unlike restriction fragment
length polymorphic (RFLP) and RAPD markers, which most
often arise from single- or low-copy sequence regions of the
genome, AFLP markers are more often derived from repeti
-
tive DNA sequences (Reaman-Buttner et al. 1999). Approxi
-
mately 77% of the total nuclear DNA in cultivated tobacco
is composed of repetitive sequences (Narayan 1987), the ma
-
jority of which appears to have been conserved during the
process of molecular evolution in this genus (Volkov et al.
1999a). Nonrepetitive DNA, which includes a majority of
the structural genes, makes up only a very small fraction of
the total tobacco genome, but should be responsible for most
of the variation in morphology, growth characteristics, bio
-
chemical composition, and quality traits found among acces
-
sions. Therefore, it might be predicted that AFLP analysis
would not be particularly effective in analyzing polymor
-
phism at the subspecies level in Nicotiana. Our experience
and data suggest otherwise. Approximately one quarter of
© 2001 NRC Canada
Ren and Timko 565
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
0
0.794 0
0.304 0.774 0
0.375 0.771 0.213 0
0.539 0.64 0.593 0.59 0
0.718 0.635 0.549 0.621 0.603 0
0.654 0.797 0.672 0.669 0.614 0.571 0
0.647 1.054 0.519 0.553 0.717 0.676 0.724 0
0.431 0.832 0.376 0.447 0.539 0.568 0.58 0.61 0
0.541 0.908 0.522 0.556 0.648 0.681 0.916 0.72 0.431 0
0.64 0.67 0.547 0.544 0.453 0.632 0.681 0.748 0.64 0.64 0
0.562 0.932 0.617 0.578 0.67 0.817 0.714 0.669 0.673 0.673 0.625 0
4.665 5.3 4.794 4.659 5.074 5.073 5.021 4.361 4.828 4.773 4.821 4.835 0
8.898 8.717 8.772 8.932 8.911 8.866 8.884 9.251 8.898 8.972 9.03 8.863 16.69 0
6.837 6.468 6.919 6.696 6.615 6.838 6.694 6.959 6.837 7.147 6.797 6.797 11.6 8.199 0
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the fragments amplified from the various accession lines of
cultivated tobacco were polymorphic and provided sufficient
resolution to distinguish among closely related cultivars and
allow six distinct subgroups to be recognized among the ac
-
cessions. Interestingly, our AFLP analysis indicates that ac
-
cessions from different geographic locations with similar
manufacturing quality traits tend to cluster based upon their
AFLP profiles, supporting the idea that such traits have a ge
-
netically definable basis.
It is worth noting that, compared with the large differ
-
ences in morphology, growth characteristics, and manufac
-
turing quality traits recognized among accession lines, the
measured extent of polymorphism is relatively small. Differ
-
ences in morphology and growth characteristics of Nicotiana
spp. in culture have been correlated previously with low lev
-
els of heterogeneity at the molecular level (e.g., differences
in RAPD profiles, Bogani et al. 1997; changes in methylation
patterns of genes in cultured cells during regeneration,
Bogani et al. 1985, 1995; Durante et al. 1986). However,
with the exception of inheritance studies of alkaloid con
-
tents, no data are available that show a direct association be
-
tween manufacturing quality traits (e.g., wrapper quality,
filler quality) and defined physiological or molecular mark
-
ers. In this regard, our AFLP data may provide a starting
© 2001 NRC Canada
566 Genome Vol. 44, 2001
Fig. 2. Dendrogram showing the phenetic relationships among 46 different accession lines of cultivated tobacco (Nicotiana tabacum).
Cluster analysis was performed using UPGMA, and bootstrap values (given at the branch points) were generated using PAUP 4.0b. In
-
cluded in the analysis are the three proposed wild progenitor species (Nicotiana sylvestris, N. otophora, and N. tomentosiformis).
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point for the more detailed analysis of the underlying ge
-
netic basis for these characteristics.
Genus Nicotiana has been divided into three subgenera,
Petunioides, Rustica, and Tabacum (Goodspeed 1954). The
eight species included in the present study are representative
of these three subgenera. In contrast with the low levels of
genetic variation we observed among cultivated N. tabacum
accession lines, interspecific variation was significantly higher.
This is evidenced by the larger D values derived from com
-
parisons of AFLP profiles of cultivated accession lines and
wild Nicotiana spp. The high degree of genetic polymor
-
phism among species that was observed using AFLP analy
-
sis is consistent with the observations of Bogani et al. (1997)
following their analysis of interspecific variation using RAPD
analysis. In addition to detecting similar levels of interspecific
variation, both molecular approaches gave very similar
phylogenetic groupings and these groupings were consistent
© 2001 NRC Canada
Ren and Timko 567
Fig. 3. Plot of scores from principal component analysis based
on AFLP profiles of cultivated accession of Nicotiana tabaccum.
Panels A and B were resolved using primer combinations of E-
ACG + M-CTT and E-AAC + M-CGT, respectively. Samples
within each of the circled clusters share similar manufacturing
quality traits. Traits are as follows: O, oriental/semi-oriental; C,
cigar wrapper/filler/binder; F, flue-cured; A, air-cured; M, Mary
-
land; B, Burley; and U, other. Prin1 and Prin 2 refer to the first
and second principal components, respectively.
Fig. 4. AFLP profiles showing the genetic polymorphism among
wild Nicotiana spp., detected using the selective primer combina
-
tions E-AGC + M-CAG (left panel) and E-AGC + M-CCC (right
panel). Lane 1, N. sylvestris; lane 2, N. otophora; lane 3,
N. tomentosiformis; lane 4, N. glutinosa; lane 5, N. suaveolens;
lane 6, N. rustica; lane 7, N. longiflora. The arrows indicate the
position of AFLP bands present in all species within genus
Nicotiana.
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with the proposed clustering of species based on traditional
analyses of cytological and morphological characteristics
(Goodspeed 1954; Gerstel 1960, 1963; Narayan 1987). For
example, Goodspeed (1954) has postulated that the present
day assemblage of species is derived from a pregeneric ge
-
netic reservoir with three major components that have been
designated pre-Nicotiana, pre-Cestrum, and pre-Petunia.
The cestroid complex is thought to be ancestral to the sub
-
genera Tabacum and Rustica and the petunioid complex to
subgenus Petunioides. Based upon the genetic distances (Ta
-
ble 3) and dendrogram (Fig. 2) generated by analysis of
AFLP data, we see N. tomentosiformis, N. otophora, and
N. glutinosa (subgenus Tabacum) forming one cluster, with
N. rustica (subgenus Rustica) being closely allied, and
N. sylvestris, N. longiflora, and N. suaveolens (subgenus
Petunioides) forming a separate cluster. Interestingly, AFLP
markers that are present in all members of the three subgenera
examined here were identified, as well as markers associated
with specific subgenera. These genus- and subgenus-specific
markers represent highly conserved sequences that have re
-
mained unaltered throughout the course of evolution and
may be useful in subsequent taxonomic, speciation, or evo
-
lution studies.
Genus Nicotiana is thought to be between 75 and 100 mil
-
lion years old (Uchiyama et al. 1977). When in its evolution
-
ary history the hybridization and polyploidization events lead
-
ing to the formation of N. tabacum, cultivated tobacco, oc
-
curred is not certain. Goodspeed (1954) and Gerstel (1960,
1963) proposed that N. tabacum, a natural allotetraploid,
originated by artificial synthesis of two ancestral genomes,
N. sylvestris (2n =2x = 24) and either N. otophora (2n =
2x = 24) or N. tomentosiformis (2n =2x = 24). These
proposals considered primarily cytological evidence and mor-
phological and growth characteristics. Based on investiga-
tions of the structure and organization of mitochondrial and
chloroplast genomes, Bland et al. (1985) and Olmstead and
Palmer (1991) demonstrated that a species similar to
N. sylvestris donated the maternal genome of tobacco. How-
ever, the paternal parent in the cross was not determined.
Kuhrová et al. (1991) found that two middle-repetitive DNA
sequences in the nuclear tobacco genome were homologous
between N. tabacum and N. tomentosiformis, implicating
N. tomentosiformis as a possible paternal donor. Similarly,
Volkov et al. (1999b) showed that intergenic spacer (IGS) re-
gions could be used as markers, to trace the molecular evo
-
lution of parental rDNA in the genome of tobacco. Their
results indicated that both N. tomentosiformis and N. sylvestris
contributed separately to the alloploid genome of N. tabacum.
The majority of tobacco rDNA repeats originated from
N. tomentosiformis, with the restructuring of the IGS occur
-
ring over time. Lim et al. (2000b) also suggested that the
majority of the genes present in N. tabacum were contrib
-
uted by N. sylvestris, with the remainder being contributed
by N. tomentosiformis. Both groups of investigators suggested
that molecular rearrangement – gene conversion played an
important role in determining the structure of repeat regions
in the N. tabacum genome and suggested that elimination
and (or) maintenance of repeat structure was a significant
factor in tobacco-genome evolution.
It should be noted that, while most investigations focused
on the involvement of N. tomentosiformis, evidence suggest
-
ing other species as possible parental donors has also sur
-
faced. For example, Kenton et al. (1993) used nonradioactive
in situ hybridization to examine the possible origin of indi
-
vidual chromosomes within cultivated tobacco. Their studies
revealed cytological evidence indicating that a part of the
tobacco genome originated from N. otophora, leading to
the suggestion that one paternal genome of tobacco may be de
-
rived from an introgressive hybrid between N. tomentosiformis
© 2001 NRC Canada
568 Genome Vol. 44, 2001
No. Species 1 2 3 4 5 6 7
1 N. sylvestris –
2 N. otophora 0.1571 –
3 N. tomentosiformis 0.1620 0.0903 –
4 N. gluutinosa 0.1915 0.1474 0.1385 –
5 N. suaveolens 0.1330 0.2035 0.1711 0.1944 –
6 N. longiflora 0.1890 0.1725 0.1793 0.1804 0.2400 –
7 N. rustica 0.1301 0.1917 0.1852 0.1852 0.1417 0.2443 –
Note: Numbers in the first row of the table are equivalent to those in the first column indicating species names.
Table 4. Genetic distance between wild Nicotiana spp., based on AFLP-profile analysis.
Fig. 5. Dendrogram showing the phenetic relationships among
seven different wild Nicotiana spp. Cluster analysis was per
-
formed using the neighbor-joining method, and bootstrap values
(given at the branch points) were generated using PAUP 4.0b.
The wild-species used in the analysis were N. sylvestris,
N. tomentosiformis, N. otophora, N. glutinosa, N. suaveolens,
N. rustica, and N. longiflora.
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and N. otophora. Support for this hypothesis comes from re
-
cent analysis of the organization and structure of the gene
family encoding putrescine N-methyltransferase (PMT)
(Hashimoto et al. 1998; Riechers and Timko 1999). Compar
-
ison of the nucleotide and deduced amino acid sequences of
the PMT gene between tobacco and its putative three ances
-
tors (N. sylvestris, N. tomentosiformis, and N. otophora)in
-
dicated that three of five PMT genes originated from
N. sylvestris, whereas of the other two, one was similar to
PMT genes present in the N. tomentosiformis genome and
one to genes present in the N. otophora genome.
Previous researchers have reported almost no single-copy
DNA divergence between N. tabacum and its diploid progeni
-
tors (Okamuro et al. 1985; Kovarik et al. 1996; Matyášek et
al. 1997; Reaman-Buttner et al. 1999; Volkov et al. 1999a).
In contrast, comparisons of differences in AFLP profiles
among N. tabacum and its three proposed progenitor species
N. sylvestris, N. tomentosiformis, and N. otophora were ex
-
tremely informative. The genetic distances shown in Table 3
and the dendrogram (Fig. 2) generated by UPGMA or
neighbor-joining analysis show a high degree of similarity
between N. tabacum and its three wild species. More impor
-
tantly, almost all the AFLP fragments present in N. tabacum
are represented in one of three putative progenitor species,
thus providing direct molecular evidence that all three wild
species contributed genetic information to N. tabacum. This
result is consistent with the cytogenetic analysis of Kenton
et al. (1993) and molecular studies of Hashimoto et al. (1998)
and Riechers and Timko (1999). However, one cannot rule
out the possibility that evolutionary loss or retention of ances
-
© 2001 NRC Canada
Ren and Timko 569
Fig. 6. AFLP profiles of 22 different accession lines of cultivated tobacco and of the three proposed wild progenitor species
(N. sylvestris, N. otophora, and N. tomentosiformis). The profiles were generated using the selective primer combination E-ACC + M-
CAC; the arrows indicate the locations of the most highly conserved AFLP markers in the profiles of the 22 cultivated tobacco acces
-
sion lines and three wild Nicotiana spp.
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tral alleles could explain the distributions of alleles observed
in these studies rather than the introgression of N. otophora
DNA, since it has been noted previously that the genomes of
allotetraploids show a tendency toward rearrangement and
loss of genetic information from one or both parental species
(Volkov et al. 1999a). Interestingly, Sinclair et al. (2000) re
-
cently reported that genetic information encoding quinolinate
phosphoribosyltransferase (QPRTase) from N. sylvestris was
missing in the genome of N. tabacum cv. NC95 but present
in other N. tabacum varieties. These data suggest that loss of
the N. sylvestris QPRTase from the N. tabacum cv. NC95
genome might have been associated with the breeding pro
-
gram leading to its development.
Acknowledgements
The authors thank Dr. Verne A. Sisson for providing seeds
of the various cultivated and wild Nicotiana spp. used in this
study. We are also grateful to Douglas Taylor, Leslie Rissler,
and Sherri Church, Christopher Botanga for their assistance
with data analysis. This research was supported by a grant to
M.P.T. from Philip Morris, Inc. (Richmond, Va.).
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