A microsatellite marker based linkage map of tobacco

Abstract

We report the first linkage map of tobacco (Nicotiana tabacum L.) generated through microsatellite markers. The microsatellite markers were predominantly derived from genomic sequences of the Tobacco Genome Initiative (TGI) through bioinformatics screening for microsatellite motives. A total of 684 primer pairs were screened for functionality in a panel of 16 tobacco lines. Of those, 637 primer pairs were functional. Potential parents for mapping populations were evaluated for their polymorphism level through genetic similarity analysis. The similarity analysis revealed that the known groups of tobacco varieties (Burley, Flue-cured, Oriental and Dark) form distinct clusters. A mapping population, based on a cross between varieties Hicks Broad Leaf and Red Russian, and consisting of 186 F2 individuals, was selected for mapping. A total of 282 functional microsatellite markers were polymorphic in this population and 293 loci could be mapped together with the morphological trait flower color. Twenty-four tentative linkage groups spanning 1,920 cM could be identified. This map will provide the basis for the genetic mapping of traits in tobacco and for further analyses of the tobacco genome.

Full text

Theor Appl Genet (2007) 114:341–349
DOI 10.1007/s00122-006-0437-5
123
ORIGINAL PAPER
A microsatellite marker based linkage map of tobacco
Gregor Bindler · Rutger van der Hoeven ·
Irfan Gunduz · Jörg Plieske · Martin Ganal ·
Luca Rossi · Ferruccio Gadani · Paolo Donini
Received: 16 May 2006 / Accepted: 17 October 2006 / Published online: 7 November 2006
© Springer-Verlag 2006
Abstract We report the Wrst linkage map of tobacco
(Nicotiana tabacum L.) generated through microsatel-
lite markers. The microsatellite markers were predom-
inantly derived from genomic sequences of the
Tobacco Genome Initiative (TGI) through bioinfor-
matics screening for microsatellite motives. A total of
684 primer pairs were screened for functionality in a
panel of 16 tobacco lines. Of those, 637 primer pairs
were functional. Potential parents for mapping popula-
tions were evaluated for their polymorphism level
through genetic similarity analysis. The similarity anal-
ysis revealed that the known groups of tobacco varie-
ties (Burley, Flue-cured, Oriental and Dark) form
distinct clusters. A mapping population, based on a
cross between varieties Hicks Broad Leaf and Red
Russian, and consisting of 186 F2 individuals, was
selected for mapping. A total of 282 functional
microsatellite markers were polymorphic in this popu-
lation and 293 loci could be mapped together with the
morphological trait Xower color. Twenty-four tentative
linkage groups spanning 1,920 cM could be identiWed.
This map will provide the basis for the genetic mapping
of traits in tobacco and for further analyses of the
tobacco genome.
Introduction
The Solanaceae are one of the most complex and
diverse plant families. Recently, considerable eVorts
have been made to catalog the biological diversity in
this plant family. With important vegetable species
such as tomato, potato, eggplants and pepper, as well
as other important plants such as tobacco and petunia,
it is economically the third most important plant taxon
(http://www.apps.fao.org). Furthermore, it has recently
been shown that coVee, a species valued as one of the
world’s most valuable agricultural commodities, is also
very closely related to the Solanaceae with respect to
its gene reservoir and genome structure (Lin et al.
2005).
Most Solanaceae are characterized by a basic chro-
mosome number of x = 12 albeit having a varying
genome size (Arumuganathan and Earle 1991) ranging
from less than 950 Mb (tomato) to approximately
3,000 Mb (pepper). Extensive genetic analyses with
molecular markers have been performed in the Solana-
ceae (Mueller et al. 2005; http://www.sgn.cornell.edu)
and in comparison with other plants such as Arabidop-
sis thaliana (Fulton et al. 2002). The genome structure
of tomato is one of the best characterized in the plant
kingdom and eVorts are underway to sequence the
Communicated by A. Schulman.
Electronic supplementary material Supplementary material is
available in the online version of this article at http://dx.doi.org/
10.1007/s00122-006-0437-5 and is accessible for authorized users.
G. Bindler · R. van der Hoeven · L. Rossi · F. Gadani ·
P. Donini (&)
Applied Research Department, Philip Morris International,
R&D, Quai Jeanrenaud 56, 2000 Neuchâtel, Switzerland
e-mail: paolo.donini@pmintl.com
I. Gunduz
Philip Morris USA, Research Center,
Richmond, VA 23261, USA
J. Plieske · M. Ganal
TraitGenetics GmbH, Am Schwabeplan 1b,
06466 Gatersleben, Germany

342 Theor Appl Genet (2007) 114:341–349
123
euchromatic area of the tomato genome in a systematic
manner. Comparative genetic mapping within the
Solanaceae has demonstrated that, e.g., the genomes of
tomato and potato diVer by a small number of inver-
sions (Tanksley et al. 1992). The genetic maps of pep-
per and eggplant diVer from tomato by a larger number
of rearrangements but nevertheless very large chromo-
somal sections have been conserved between these
species (Livingstone et al. 1999; Doganlar et al. 2002a,
b).
The genus Nicotiana (Goodspeed 1954) is a well-
deWned group of species of which tobacco (Nicotiana
tabacum L.) is an important agricultural crop plant
that plays a signiWcant role in the economies of many
countries (Davis and Nielsen 1999; http://www.fao.org/
documents/show_cdr.asp?url_Wle=/DOCREP/006/Y49
97E/ Y4997E00.HTM).
Furthermore, tobacco is considered to be one of the
most important model systems in plant biotechnology.
Being an easily transformable plant, tobacco serves the
role of an experimental system that is frequently being
used for pilot studies on the expression of novel
transgenes that are later being used in important food
crops. Through its high level of biomass accumulation,
tobacco is a species that is considered to be highly
promising for the production of commercially impor-
tant substances (e.g., medical drugs and vaccines) in
plants. Furthermore, Nicotiana species are investigated
for aspects concerning the elucidation of principles of
disease resistance, synthesis of secondary metabolites
and basic questions of plant physiology.
Albeit an inbreeding, highly homozygous plant, as
well as being of high economic and scientiWc impor-
tance, it is surprising that the genetic analysis of
tobacco is still in its infancy. While detailed linkage
maps are available for several solanaceous plants, the
genetic mapping of tobacco has not made any signiW-
cant progress in the last 15 years except for a genetic
map in a population derived from a cross between the
diploid Nicotiana plumbaginifolia and Nicotiana longi-
Xora (Lin et al. 2001). Only punctual molecular marker
analyses have been performed in allotetraploid
tobacco (Nicotiana tabacum) such as the analysis of
genetic relationships in wild and cultivated tobacco
material (Bogani et al. 1997; Ren and Timko 2001;
Rossi et al. 2001) or the tagging of individual disease
resistance genes with molecular markers such as
RAPDs and AFLPs (Nishi et al. 2003; Julio et al.
2005).
Furthermore, there is basically no information
available regarding the relationship of the individual
chromosomes (synteny) with those of other Solanaceae
albeit many of these plants share the same basic
chromosome number. Reasons for this are most likely
that Nicotiana tabacum is a tetraploid species. Tobacco
is considered to be an allopolyploid interspeciWc hybrid
between Nicotiana sylvestris (n = 12) and Nicotiana
tomentosiformis (n = 12) and has n = 24 chromosomes
(Kenton et al. 1993; Lim et al. 2004). Another reason
for the lack of a detailed genetic analysis of the tobacco
genome is possibly that the tobacco genome is with
approximately 4,500 Mbp at the high end of the
genome size in the Solanaceae (Arumuganathan and
Earle 1991). As a consequence of this large genome
size, a large proportion of the tobacco genome is repre-
sented by highly repeated DNA sequences including
rDNA (Borisjuk et al. 1997; Volkov et al. 1999), active
transposons (Casacuberta et al. 1995) and retrotrans-
posons (Gregor et al. 2004).
Only recently, through the eVorts of the Tobacco
Genome Initiative (TGI, http://www.tobaccoge-
nome.org) eVorts have been undertaken regarding a
systematic characterization of the tobacco genome
(Gadani et al. 2003). The aim of the TGI is the identiW-
cation of more than 90% of all tobacco genes through a
combination of EST sequencing, the sequencing of
undermethylated low copy DNA and the generation of
BAC libraries for physical mapping. Considerable pro-
gress has been made since the initiation of TGI but this
project lacks until now a genetic component compris-
ing the construction of a high-resolution genetic map
that could provide a framework for anchoring the
sequencing information to the tobacco chromosomes.
The aim of the research reported in this paper is the
construction of a Wrst draft of a tobacco genetic map
which could serve as genetic backbone for the integra-
tion of the sequence data generated from the TGI
while constituting a Wrst step towards a comparative
genome analysis with other solanaceous species.
Materials and methods
Plant material and DNA isolation
All plants were grown from seeds in the greenhouse.
For the analysis of polymorphism a panel of 16 tobacco
varieties (Table 1) representing the main types of
tobacco (Flue-cured, Burley, Oriental and Dark/oth-
ers) was used. For each variety, leaves from a pool of 6
plants were combined for DNA extraction. The map-
ping population (kindly provided by Prof. Ramsey
Lewis, North Carolina State University) was derived
from a cross between the varieties Red Russian and
Hicks Broad Leaf (a Flue-cured tobacco type). The F2
population consisted of 186 individual plants that were

Theor Appl Genet (2007) 114:341–349 343
123
grown to maturity in a greenhouse. Total genomic
DNA was isolated according to the protocol of Rogers
and Bendich (1988) from leaf tissue.
PCR conditions and allele detection
PCR was performed in 10 l volumes containing
approximately 25–50 ng of template DNA, 1 £ PCR
buVer (50 mM KCl, 10 mM Tris–HCl pH 8.3), 1.5 mM
MgCl
2
, 0.15 mM of each primer, 0.25 mM dNTPs and
0.3U of Taq polymerase (Applied Biosystems). Ther-
mocycling started with a denaturation step for 3 min at
94°C followed by 45 cycles of 1 min at 94°C, 1 min at
55°C, 2 min at 72°C and stopped after a Wnal extension
step of 72°C for 7 min. The fragment analysis was per-
formed as a multi-loading assay analyzing two or three
markers simultaneously that were labeled by diVerent
ABI-dyes. Samples containing 0.5–1 l PCR products
of each marker, 1 l internal size standard and 9 l Hi-
Di formamide were separated using 36 cm capillary
arrays. Alleles were detected using the GeneScan/
Genotyper
®
software package of Applied Biosystems.
Molecular markers
A total of 187 SSR markers had previously been
developed from a microsatellite-enriched genomic
library of the Burley tobacco variety TN90 (Philip
Morris International, unpublished data). Further
microsatellite identiWcation and marker develop-
ment employed data from the TGI (http://www.
tobaccogenome.org). A pre-screen on approximately
50,000 non-redundant methyl Wltered tobacco
sequences was performed to identify tandem repeats
using the program Tandem Repeat Finder (Benson
1999). The program settings permitted the identiWca-
tion of di-, tri- (ten repeat units minimum), tetra-,
hexa- and penta-nucleotide repeats (Wve repeat units
minimum) with at least 80 Xanking bases. Then, the
resulting sequences were used to identify microsatel-
lite containing DNA sequences as candidate target
sites for development of SSR markers. The
sequences were received as multiple fasta Wles with
the corresponding quality Wles. Subsequently, vector-,
E. coli- and duplicated sequences were masked in the
microsatellite containing sequences by using the
cross_match
©
program (University of Washington,
distributed by CodonCode Inc.). Following a trans-
formation into experimental sequencing Wles, di- to
penta-nucleotide microsatellite containing sequences
were selected with a minimum of 20 bp length and a
minimum match of 90% of the virtual microsatellite
motif. Oligonucleotide primer pairs Xanking the micro-
satellite sequence were designed using the Primer
3.0 program (Steve Rozen and Helen J. Skaletsky,
Whitehead Institute for Biomedical Research). Prim-
ers were selected to be approximately 20 bp long, to
have a GC content between 20 and 80%, and with a
melting temperature between 57 and 63°C (optimum
60°C). Forward primers were labeled with either
FAM, HEX or ROX for fragment analysis on Applied
Biosystems 3100 Genetic Analyzers. Primer sequences
are available from the electronic supplementary
material (S1).
Genetic mapping
The genetic mapping of polymorphic markers was per-
formed using the JoinMap
®
3.0 program (Van Ooijen
and Voorrips 2001) with the following settings: used
linkages with REC smaller than 0.400 and LOD larger
than 1.00, threshold for removal of loci with respect to
jumps in goodness-of-Wt 5.000, mapping function Kos-
ambi. Flower color was scored visually in all F2 plants
and integrated into the genetic map as a dominant
trait.
Cluster analysis
The genetic relationship of the 16 tobacco varieties
was investigated using the NTSYSpc 2.11j program
(Exeter Software, Setauket, NY 11733–2870) with
the following settings: Qualitative data, Dice coeY-
cient (Dice 1945), Sequential Agglomerative Hierar-
chical and Nested clustering method (SAHN),
Unweighted Pair-Group Method, Arithmetic average
(UPGMA).
Table
1
L
i
st o
f
to
b
acco var
i
et
i
es use
d
f
or test
i
ng t
h
e var
i
a
bili
ty
of the tobacco microsatellite markers
No. Variety
NT0001 OR Izmir Ege 64
NT0002 OR Prilep 12 2/1
NT0003 OR Basma Xanthi
NT0004 OR Samsum
NT0005 BU TN90
NT0006 BU TN86
NT0007 BU Kentucky 14
NT0008 BU Banket A1
NT0009 FC K326
NT0010 FC K346
NT0011 FC Kutsaga 35
NT0012 FC Hicks Broad Leaf
NT0013 Dark Dac Mata Fina
NT0014 Dark Amarillo Parado
NT0015 Dark Criollo Misionero
NT0016 Red Russian

344 Theor Appl Genet (2007) 114:341–349
123
Results
Marker development
The microsatellite markers employed in this study
were derived from two diVerent sources. One set of
187 markers (PT1... and PT2...) has been developed
previously (Philip Morris International, unpublished
data). The majority and second set of markers (PT3...
and PT4...) was generated from screening the TGI
sequences for di- to hexanucleotide motives using bio-
informatic means. Five hundred such microsatellite
motifs containing sequences were used for subsequent
primer development. A total of 684 primer pairs were
tested on the panel of 16 lines. After the analysis on
the 16 lines, markers were deWned as either functional
or non-functional. Functional markers were deWned by
a maximum of six ampliWcation fragments, with at
least one of the fragments being precisely in the
expected size range as deduced from the genomic
sequence. Non-functional were those markers for
which ampliWcation failed in all the lines, or resulted in
ampliWcation of more than six fragments, or the
marker showed an excess of stuttering on the chro-
matogram. The majority of the functional markers
(77%) ampliWed either one or two fragments in the
tobacco genome. Figure 2 shows examples of two
high-quality markers that amplify either one or two
loci from the tobacco genome. AmpliWcation of one
fragment can be interpreted as a primer pair that binds
speciWcally to only one of the two genomes of tobacco
and not to the other while two fragments are ampliWed
when a primer pair binds to the (probably homolo-
gous) region in the two diVerent genomes that consti-
tute the allotetroploid genome of Nicotiana tabacum.
Table 3 shows a summary of the results for the 684
investigated microsatellite markers regarding their
functionality, number of ampliWed fragments and
polymorphism in the actual mapping population
whereby no signiWcant diVerences were observed
between the two diVerent sources.
Parental survey and selection of mapping population
One of the prerequisites for eYcient genetic mapping
in a given plant species is the availability of a highly
polymorphic mapping population. For this, a set of 16
selected varieties/lines was screened to investigate the
level of polymorphisms between potential parents for
the mapping population. This set of lines comprised of
diVerent tobacco types such as Flue-cured, Burley, Ori-
ental and Dark/other tobaccos. A set of 90 high-quality
microsatellite markers out of all tested functional
microsatellite markers was used for the evaluation of
potential parents. The results of the study are shown in
Table 2 and Fig. 1. The genetic similarity coeYcient
within these samples ranged from a very low value of
0.212 for the combination of Oriental Prilep and Bur-
ley Banket to a very high value of 0.918 for the combi-
nation of Flue-cured tobacco line K326 and Flue-cured
tobacco line K346 indicating that a wide range of
genetic diversity was present in these selected 16 sam-
ples. Furthermore, a cluster analysis for these 16 lines
shows that the various tobacco types clustered together
Fig. 1 Cluster analysis of the
16 tobacco varieties based on
the data of 90 polymorphic
markers using the similarity
coeYcient of Dice (1945).
OR = Oriental; BU = Burley;
FC = Flue-cured
Coefficient
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
Prilep12-2/1MW
IzmirEge64
Prilep12-2/1
BasmaXanthi
Samsum
RedRussian
DarkCriollo
TN90
TN86
Kentucky14
BanketAl
K326
K346
Kutsaga35
Hicks
DarkDacMata
DarkAmarillo
OR
BU
FC

Theor Appl Genet (2007) 114:341–349 345
123
in almost all cases. The three main tobacco types Bur-
ley, Flue-cured and Oriental formed three distinct
groups with deWned clusters. Only the Dark tobacco
types did not cluster in a distinct group. In no case,
however, Dark tobaccos were found to group within a
cluster of the other tobacco types, indicating that the
three groups of Burley, Flue-cured and Oriental tobac-
cos were clearly genetically distinct.
Generation of a tobacco genetic map
with microsatellite markers
Based on the availability of mapping populations; the
cross between the Flue-cured variety Hicks Broad
Leaf and Red Russian was chosen as a mapping popula-
tion for the generation of a genetic map. The genetic
similarity coeYcient between these two lines was 0.385
Fig. 2 Examples for microsatellite patterns in the investigated 16
tobacco varieties. a Dinucleotide repeat containing marker with
slight stutter that detects one locus on one of the tobacco ge-
nomes and is polymorphic between the parents of the mapping
population. b Dinucleotide repeat containing marker with slight
stutter that detects two loci. One of the two loci is polymorphic
between the parents of the mapping population

346 Theor Appl Genet (2007) 114:341–349
123
which is close to the low end of all pair-wise combina-
tions. For mapping, a total of 186 F2 plants were used.
Mapping data could be generated for a total of 282 poly-
morphic microsatellite markers. More than 50% of the
mapped markers ampliWed only a single fragment
(Table 3) which is indicative for ampliWcation from only
one of the two tobacco genomes. Approximately, one
quarter of the mapped markers ampliWed two fragments
in the tobacco genome suggesting that one fragment is
being ampliWed from each of the two tobacco genomes.
However, in nearly all cases, for markers amplifying two
fragments, only one fragment could be mapped due to
the fact that the other fragment was monomorphic in
the mapping parents and frequently also in the other
panel plants, so that the total number of mapped loci
added up to only 293. The resulting genetic map of
tobacco is presented in Fig. 3. A total of 24 linkage
groups could be identiWed, although three putative link-
age groups (3a/3b, 8a/8b and 14a/14b) were each still
divided into two sub-linkage groups of markers each,
whereby JoinMap, however, suggested that they should
group together. At present, Wve markers cannot be
linked to any of the identiWed linkage groups, indicating
that the genetic map is not yet covering the entire length
of the tobacco genome. The entire map covers approxi-
mately 1,930 cM. Since tobacco is an allotetraploid spe-
cies, the genetic length of each genome is in the present
map close to 1,000 cM. The morphological trait of
Xower color could be integrated into linkage group 5.
Discussion
Microsatellite markers as markers for mapping
allopolyploid plants
Microsatellite markers have been used for the genera-
tion of a molecular linkage map of tobacco. The gen-
eral advantages of microsatellite markers for the
generation of molecular linkage maps and marker
analyses in plants have been reviewed extensively. In
addition to these general advantages, microsatellite
markers are currently the marker system of choice for
the genetic analysis of allopolyploid plant species due
to several characteristics.
First, microsatellite markers are usually derived
from genomic sequences residing outside genes.
Because of the high variability of such non-coding
sequences, a large number of markers amplify only one
fragment. For example, in the allohexaploid wheat
genome approximately 40% of the developed micro-
satellite markers are genome speciWc (Röder et al.
1998). In the allotetraploid, Brassica napus 30% of all
markers are genome-speciWc (Plieske and Ganal,
unpublished results) and in tobacco nearly 60% of all
markers amplify only a single fragment from one of the
Table 2 Similarity table of the 16 investigated tobacco varieties based on data for 90 microsatellite markers, indicated as similarity
coeYcient of Dice (1945)
NT01 NT02 NT03 NT04 NT05 NT06 NT07 NT08 NT09 NT10 NT11 NT12 NT13 NT14 NT15 NT16
NT01 1.000
NT02 0.459 1.000
NT03 0.415 0.606 1.000
NT04 0.379 0.606 0.595 1.000
NT05 0.313 0.246 0.325 0.260 1.000
NT06 0.337 0.251 0.346 0.300 0.918 1.000
NT07 0.298 0.304 0.358 0.300 0.727 0.726 1.000
NT08 0.328 0.212 0.316 0.245 0.876 0.853 0.695 1.000
NT09 0.344 0.276 0.302 0.312 0.513 0.505 0.500 0.423 1.000
NT10 0.352 0.274 0.290 0.277 0.528 0.497 0.516 0.449 0.902 1.000
NT11 0.341 0.311 0.353 0.378 0.556 0.534 0.516 0.545 0.636 0.658 1.000
NT12 0.342 0.235 0.294 0.333 0.545 0.507 0.525 0.495 0.719 0.694 0.774 1.000
NT13 0.393 0.436 0.432 0.370 0.405 0.424 0.435 0.382 0.437 0.433 0.402 0.373 1.000
NT14 0.332 0.367 0.380 0.309 0.419 0.431 0.428 0.381 0.448 0.433 0.479 0.458 0.437 1.000
NT15 0.289 0.306 0.333 0.284 0.262 0.302 0.273 0.299 0.276 0.249 0.324 0.267 0.333 0.392 1.000
NT16 0.372 0.376 0.505 0.352 0.355 0.351 0.363 0.330 0.373 0.382 0.396 0.385 0.419 0.434 0.346 1.000
Table 3 Functionality and number of ampliWed loci of the inves-
tigated tobacco microsatellite markers
All Percentage Mapped Percentage
1 locus 357 52.2 165 58.5
2 loci 171 25.0 82 29.1
3 loci 21 3.1 12 4.3
4 loci 5 0.7 4 1.4
Multiple loci
(<7)
83 12.1 19 6.7
Non-functional 47 6.9 0 0
684 100 282 100

Theor Appl Genet (2007) 114:341–349 347
123
two genomes. This feature permits the assignment of
markers, linkages to traits and linkage maps to speciWc
chromosomes in a more reliable way than with other
marker systems. Microsatellite markers that amplify a
single fragment are the most suitable markers for the
analysis of genetic relationships in allopolyploid spe-
cies since they do not have problems with perturba-
tions in the data analysis due to loci ampliWed from
other genome(s). On the other hand, the main disad-
vantage of microsatellite markers is that they cannot be
used for synteny studies due to their genome-speciWcity
and origin from non-coding sequences since it is very
likely that they will not amplify fragments from even
closely related genomes unless they are derived from
coding sequences.
Second, due to their multiallelic structure, microsat-
ellite markers are the only reliable marker system that
can be analyzed in a predominantly codominant fash-
ion in a segregating population even in allopolyploid
species. Most other PCR-based marker systems such as
RAPDs or AFLPs can in polyploids only be analyzed
as dominant markers. The same is true for the biallelic
SNP (single nucleotide polymorphism) markers since
they are mostly derived from highly conserved genes
and, thus, are usually not genome-speciWc and only
scorable as presence/absence markers.
Genetic relationships of tobacco varieties
The data on genetic relatedness of tobacco varieties
clearly suggest that the established groups of tobacco
varieties are based on diVerent germplasm pools. The
three groups of Flue-cured, Burley and Oriental
tobacco varieties form clear clusters. The fourth group
represented by the Dark tobacco varieties does not
form a clear monophyletic group due to the fact that
the genetic relationships between the investigated lines
are very limited and thus, this cluster might represent
an ancestral group containing a large amount of
genetic variability. Previous AFLP analyses have sug-
gested a similar population structure but have not pro-
vided suYcient resolution to substantiate such a clear
distinction of the individual groups since only eight
primer combinations were used (Ren and Timko 2001;
Rossi et al. 2001). In the future, the analysis of addi-
tional tobacco lines using a set of high quality microsat-
ellite markers should provide a very detailed picture of
the genetic relatedness of tobacco lines within and
between individual groups of germplasm. Further-
more, these data show that a limited set of microsatel-
lite markers should be suYcient for variety
identiWcation and grouping of tobacco lines and varie-
ties through the use of microsatellite marker databases.
Fig. 3 Genetic map of Nicotiana tabacum consisting of 282 mi-
crosatellite markers and 293 mapped SSR loci and one pheno-
typic trait (Xower color) on 24 linkage groups. Five markers are
still unlinked. The markers of three groups (3, 8 and 14) are each
distributed on two subgroups, but probably belong to one linkage
group (LOD score > 4). If two loci were mapped with one mark-
er, the two loci were labeled a and b
PT303510.0
PT300570.8
PT203918.5
PT201898.8
PT3048527.0
PT30142b27.5
PT3025938.1
PT3030741.8
PT3042468.0
PT3038770.5
1
PT303750.0
PT201681.8
PT201638.5
PT30
327
11.
2
PT4003517.0
PT3011418.1
PT3024218.6
PT3006729.2
2
PT201720.0
PT10379.9
PT30308
PT30368
10.2
PT3027715.8
PT3014616.7
PT2028719.5
PT109521.7
3a
PT400210.0
PT301835.4
PT203725.5
PT124216.5
PT3019717.1
PT2030
6a
18.7
PT3022919.5
PT3018530.0
PT3020540.5
3b
106.0
PT203430.0
PT30
346
2.0
PT30
161
12.3
PT3012420.2
PT3002122.7
PT3047824.6
PT3035434.5
PT3001635.2
PT125035.7
PT20315
PT30172
PT30209
36.0
PT3020140.2
PT3045550.5
PT3022366.3
PT30
224
66.5
PT3015171.8
PT3027282.8
PT30304
4
PT304520.0
PT303717.3
PT2016512.0
PT3015729.6
PT2021138.4
PT3044956.2
PT3029659.4
PT3008763.7
PT2023464.0
PT3024584.3
PT3018494.8
PT1199120.0
PT30169122.5
PT30477130.2
6
PT302020.0
PT300255.0
PT303945.5
PT3013812. 3
PT30393a14. 9
PT3021525. 8
PT132
2
33.1
PT1047133. 3
PT30415c39.9
PT3041940. 9
PT3029250. 4
7
PT301740.0
PT302600.8
PT30
395
21.9
PT3021823.1
PT3014724.3
PT130049.8
PT3036662.4
PT3016776.8
PT3010779.3
PT30164100.6
PT30388100.9
PT20244107.5
PT1399108.3
PT2017
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PT3025587.2

348 Theor Appl Genet (2007) 114:341–349
123
The tobacco microsatellite map
This genetic map of Nicotiana tabacum represents the
Wrst version of a linkage map that covers most of the
tobacco genome. With 293 loci covering a genetic dis-
tance of 1,930 cM, each of the two ancestral genomes
should cover approximately 1,000 cM. It is clear that
the current version of the tobacco linkage map is not
yet complete since some unlinked markers exist as well
as some potentially large gaps in at least three linkage
groups. For a more complete map, it will be necessary
to map additional microsatellite markers onto the
tobacco linkage map. Based on the mapping experi-
ence in tomato and other diploid solanaceous plants, a
genetic map that covers the entire genome with molec-
ular markers will need at least 300–350 markers per
genome resulting in a need of an excess of 600–700
microsatellite markers to suYciently cover the two
genomes of tobacco without major gaps. It is interest-
ing to note that the total genetic distance of approxi-
mately 1,000 cM per genome is very close to the total
genetic distance that has been observed for the diploid
tomato with a similar set of markers (approx. 150), sug-
gesting that the total length of the map for each of the
two tobacco genomes might be in the range of 1,400–
1,500 cM, thus being similar to the tomato genome
(Bernatzky and Tanksley 1986; Tanksley et al. 1992).
An assignment of the individual linkage groups to
the two genomes derived from Nicotiana sylvestris and
Nicotiana tomentosiformis is not possible at present
since the ancestral genomes have not yet been included
into the microsatellite analysis. Due to the genome
speciWcity of more than 50% of the mapped microsatel-
lite markers, it will probably be possible to assign each
linkage group to one of the two genomes in the future.
Furthermore, through the availability of monosomic
alien addition lines, it will be possible to merge the
genetic and cytogenetic maps of tobacco with its two
ancestral genomes (Suen et al. 1997). Another impor-
tant point would be the identiWcation of the homeolo-
gous chromosome pairs for the sylvestris and
tomentosiformis genome of Nicotiana tabacum through
the mapping of markers on both genomes. However,
with only ten markers that ampliWed two polymorphic
fragments and could be mapped on two diVerent chro-
mosomes and considering the incompleteness of the
map, such an assignment would be speculative until
more such markers have been identiWed and mapped.
The mapping of a phenotypic trait (Xower color) to
linkage group 5 demonstrates that with this microsat-
ellite-based linkage map it is possible to localize
monogenic and possibly also polygenic traits in the
tobacco genome thus oVering scope for marker-
assisted selection of traits in the frame of marker-
assisted breeding programs. Previously, the use of
anonymous RAPD or AFLP markers has not permit-
ted such a precise mapping and chromosomal assign-
ment. Furthermore, in the long-term it will be possible
to integrate and compare the chromosomal position
and structure of Xower color genes with other Solana-
ceae (De Jong et al. 2004)
Future developments
The microsatellite marker development and analysis
reported in this paper would not have been possible
without the availability of genomic and especially sin-
gle-copy genomic sequences generated through the
sequencing of methyl-Wltrated sequences in the frame
of the TGI, since these sequences were the source of
most of the developed markers. Together with the
construction of BAC-libraries and BAC-end sequenc-
ing, it will in the future be possible to generate addi-
tional microsatellite markers for the completion and
further saturation of the tobacco genetic map to a den-
sity that is comparable to that of other solanaceous
species such as tomato, potato and pepper, with more
than 1,000 markers per genome (Tanksley et al. 1992).
Only such marker density will result in suYcient mark-
ers for the genetic analysis within deWned germplasm
pools of tobacco. Furthermore, the integration of a
large number of genetic markers into future physical
maps based on BAC-Wngerprinting will be necessary
to generate a reliable physical map of the tobacco
genome.
The TGI has also resulted in a large set of ESTs
from the tobacco genome which will permit a compari-
son of the gene repertoire of tobacco with that of
other, well characterized, solanaceous species such as
tomato, potato and pepper, as well as closely related
species outside of the Solanaceae such as coVee. Map-
ping of conserved ESTs into the tobacco genetic map
through microsatellite markers that are generated from
ESTs or through the mapping of SNPs in conserved
genes will in the long term provide a picture of the
diVerences in gene order between tobacco and other
Solanaceae and extend our knowledge concerning
genome evolution and structure in this important plant
group.
Acknowledgments Prof. Ramsey Lewis is acknowledged for
providing the seeds for the mapping population. TraitGenetics
GmbH is thankful to Anika Küttner and Doris Kriseleit for their
qualiWed technical assistance. Furthermore, the eVorts of Alec
Hayes (Philip Morris USA), C. Opperman and S. Lommel (both
North Carolina State University) within the TGI are acknowl-
edged.

Theor Appl Genet (2007) 114:341–349 349
123
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