Euphytica (2015) 202:479–486
DOI 10.1007/s10681-015-1355-x
Mapping quantitative trait loci determining seed longevity
in tobacco (Nicotiana tabacum L.)
M. Agacka-Mołdoch • M. Nagel •
T. Doroszewska • R. S. Lewis • A. Börner
Received: 30 November 2014 / Accepted: 9 January 2015 / Published online: 21 January 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract Seed longevity is a crucial issue for
germplasm conservation and seed marketing. This
trait is determined not only by environmental condi-
tions, but also by genetic factors. Molecular mapping
of responsible loci has been performed with several
crops, but not with tobacco (Nicotiana tabacum L.). In
the present study, we investigate 122 recombinant
inbred lines derived from a cross between the cultivars
Florida 301 and Hicks. Four germination-related traits
were studied by examining seeds either untreated or
after controlled deterioration (CD): total germination
(TG, %), normal germination (%), time to reach 50 %
of TG (h), and the area under the curve after 200 h of
germination. Whereas Hicks exhibited high germina-
tion percentage and speed in untreated (fresh) seeds,
Florida 301 seems to withstand the CD treatment
better, having increased seed longevity. In total, four
genomic regions located on four different linkage
M. Agacka-Mołdoch
M. Nagel
A. Börner (&)
Leibniz Institute of Plant Genetics and Crop Plant
Research, Corrensstr. 3,
06466 Stadt Seeland, OT Gatersleben, Germany
e-mail: boerner@ipk-gatersleben.de
M. Agacka-Mołdoch
T. Doroszewska
Institute of Soil Science and Plant Cultivation, State
Research Institute, ul. Czartoryskich 8, 24-100 Puławy,
Poland
R. S. Lewis
Department of Crop Science, North Carolina State
University, Campus Box 7620, Raleigh, NC 27695, USA
groups (LGs) were identified to be associated with the
selected traits. Positive alleles for the individual traits
were contributed by both parents. A major quantitative
trait locus (QTL) for high percentage TG located on
LG 8/18 appeared in both control and deteriorated
seeds and was contributed by Hicks. In contrast,
Florida 301 donated a favorable allele for germination
speed on LG seven after CD only. The position of this
locus compared well with a QTL detected in the same
population, in a former study examining resistance
against the black shank disease caused by Phytoph-
thora nicotianae). The effects of environmental
growing conditions of the mother plants on seed
longevity are discussed.
Keywords Controlled deterioration
Germination

QTL mapping
Storability
Introduction
The lifespan, or longevity, of seeds is an important
subject, not only for the seed industry but also for the
ex situ conservation of germplasm collections. It is
known that there are distinctive differences between
crops. The first scientific papers on crop-specific
differences in seed longevity appeared in the nine-
teenth century. Haberlandt (1873) reported that barley
lost half of its germination capacity in 4 years, maize
and oats lost half in 6 years, wheat lost practically all
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in 6 years, and rye lost all in 3 years. More recent
studies, by Walters et al. (2005) and Nagel and Börner
(2010), confirm that seed deterioration behavior is
species specific. However, there are also hints of an
intraspecific variability. Early studies on rice (Jones
1926) and soybeans (Burgess 1938) suggested that
some varieties stored better than others under compa-
rable conditions. Other examples of varietal differ-
ences in seed longevity for a range of crop species are
described by Priestley et al. (1985).
In order to study the longevity of seeds in a
reasonable period of time (not spending years) artifi-
cial ageing tests were established. Seeds are exposed
for short periods to the two environmental variables
which cause rapid seed weakening; high temperature
and high relative humidity (RH). High vigour seed lots
will withstand these extreme stress conditions and
deteriorate at a slower rate than low vigour ones
(Hampton and TeKrony 1995).
First attempts at determining the genetic loci
responsible for seed storability were made for Arabi-
dopsis (Bentsink et al. 2000) and rice (Miura et al.
2002). For Arabidopsis, the authors analyzed 162
recombinant inbred lines (RIL) of a bi-parental cross.
Four putative quantitative trait locus/loci (QTL)
affecting seed longevity were identified on three
different chromosomes. Another RIL population
comprising 114 lines was investigated by Clerkx
et al. (2004). The authors detected three QTL on three
different chromosomes, two of which were in highly
comparable positions to those detected by Bentsink
et al. (2000).
For rice, Miura et al. (2002) initiated a study on
seed longevity using 98 backcross inbred lines.
Analysis revealed three QTL. Zeng et al. (2006) and
Xue et al. (2008) analysed 127 doubled haploid lines
and 71 RILs, respectively, detecting three QTL each.
All three studies pointed to rice chromosome 9 as the
carrier of an important genomic region affecting seed
longevity in comparable chromosomal regions.
In addition to the pilot examinations in Arabidopsis
and rice, genetic studies on seed longevity also
followed for soybean (Singh et al. 2008), barley
(Nagel et al. 2009, 2014), maize (Revilla et al. 2009),
wheat (Landjeva et al. 2010; Rehman Arif et al. 2012),
lettuce (Schwember and Bradford 2010), and oilseed
rape (Nagel et al. 2011).
Comprehensive studies on seed longevity for
Nicotiana species were initiated by Agacka et al.
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Euphytica (2015) 202:479–486
(2013, 2014). Seeds stored at different temperatures
(20, 0, -15/-18 °C) were investigated. It was dem-
onstrated that decreasing the temperature from 20 to
0 °C increases the storability of seeds of selected
Nicotiana species from about 10 to 30 years, and that
decreasing it further, to -15/-18 °C, increases stor-
ability to more than 50 years, as measured by a
germination threshold higher than 75 %. As in other
species, intraspecific variation was noticeable.
In order to study the genetics of seed longevity in
Nicotiana, we followed an approach that had been
adopted for barley, wheat, and rapeseed (Nagel et al.
2009, 2011, 2014; Rehman Arif et al. 2012). Seed lots
of a previously existing mapping population, devel-
oped for detecting QTL for distinctive characters,
were used for investigating germination related traits.
In the present study, we used seeds of RILs derived
from a cross between tobacco (Nicotiana tabacum)
cultivars Florida 301 and Hicks. Originally, this
mapping population was developed with the aim of
finding QTL associated with resistance to black shank,
caused by Phytophthora nicotianae (Xiao et al. 2013).
Materials and methods
Plant material
A population of 122 RILs (F5:6) was derived from the
cross Florida 301 9 Hicks using single seed descent.
Seed of the RILs was produced from greenhouse-
grown plants in 2007, whereas seed of the parental
lines originated from field plots in 2004. Seed were
stored under ambient temperatures, but at a relative
humidity of about 30 % until experiment was carried
out in 2014. The linkage map, consisting of 339
markers distributed among 24 linkage groups (LGs),
covered a total length of 1,176.54 cM (Xiao et al.
2013). The number of markers on each LG varied from
5 to 29. LG numbers were assigned as published by
Bindler et al. (2011).
Experimental design
Seed germination for control treatment (C) and after
controlled deterioration (CD) was performed for each
entry in four replications (independent experiments)
of 50 seeds per line on moistened filter paper (90 mm
roundfilter, C 160; Munktell & FILTRAK GmbH,
Euphytica (2015) 202:479–486 481

Germany) in Petri dishes placed in a climate chamber
(Panasonic MLR-352-PE). Corresponding to ISTA
(2014) the temperatures were 30 ± 2 and 20 ± 2 °C,
during illumination (8 h) and darkness (16 h),
respectively.
In order to determine the germination speed,
germinated seeds (visible radical emergence) were
counted daily within a germination period of 10 days.
Based on these counts, the time needed to reach 50 %
of total germination (TG) and the area under the curve
(AUC; the integration of the fitted curve between
t = 0 and a defined endpoint t = x) after 200 h of
germination were determined using germination soft-
ware GERMINATOR (Joosen et al. 2010). On day 10,
the TG and the number of seedlings showing a normal
appearance [normal germination (NG)] according to
ISTA (2014) were recorded.
To achieve identical initial moisture content, seeds
were placed into permeable paper bags and equili-
brated at 22 ± 2 °C and 10 % (RH). One week before
CD seeds were placed above 8.7 M unsaturated
lithium chloride LiCl (47 % RH and 20 °C, calculated
based on the Seed Information Database, Royal
Botanic Gardens, Kew, UK) on plastic racks, which
were positioned in airtight boxes. Afterwards, unsat-
urated LiCl solution was replaced by 7.1 M LiCl
solution to reach RH of 60 % inside the boxes (Hay
et al. 2008). The boxes were placed in ageing
chambers at a temperature of 45 °C for 30 days.
Temperature and time were chosen based on a pre-
experiment performed with the parental lines only
(data not shown).
QTL analysis was conducted using software QTL
Cartographer 2.5 (Zeng 1994). QTL having LOD
scores higher than three (major QTL) were considered.
QTL having LOD[2,\3 were considered only when
they appeared in positions of major QTL. Mapping
was performed for traits (C and CD), %TG (%TG-C,
%TG-CD), %NG (%NG-C, %NG-CD), time needed
to reach 50 % of TG (T50-C, T50-CD), and AUC after
200 h of germination (AUC-C, AUC-CD).
Results
Analysis of phenotypic data
The distributions of the RILs, as well as the positions
of the parents for the germination-related traits, are
given in Figs. 1 and 2. Because the seeds of the parents
had originated from a different regeneration year, a
direct comparison with the RILs is not possible.
However, one can assess the responses of both parents
separately. Superior performance of Hicks as com-
pared to Florida 301 under control conditions was
observed for %TG and %NG (Fig. 1). After the CD,

Fig. 1 Distributions for the % TG Control % NG Control

80 80
traits % total germination
70 70
Number of Lines

Number of Lines

(%TG) and % normal 60

Florida 301 Hicks
60
Florida 301 Hicks
germination (%NG) of 50 50
untreated control seeds and 40 40
seeds after CD for RILs 30 30
derived from the Florida 20 20
10 10
301 9 Hicks cross. 0
0
Positions of the parents are
indicated by arrows
% Total Germination % Total Germination
% TG Controlled Deterioration % NG Controlled Deterioration
80 80
70 70
Number of Lines

Number of Lines

60 60
50 Florida 301 50
Florida 301
40 40
30 Hicks 30 Hicks
20 20
10 10
0 0

% Total Germination % Total Germination

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482 Euphytica (2015) 202:479–486

T50 Control AUC Control

40 40
35 35
Number of Lines

Number of Lines
Hicks Florida 301 Florida 301 Hicks
30 30
25 25
20 20
15 15
10 10
5 5
0 0

Time needed to reach 50 % germination (h) Area under the curve after 200h
T50 Controlled Deterioration AUC Controlled Deterioration
40 40
35 35
Number of Lines

Number of Lines
30 30
Florida 301 Hicks
25 25 Hicks Florida 301
20 20
15 15
10 10
5 5
0 0

Time needed to reach 50 % germination (h) Area under the curve after 200h

Fig. 2 Distributions for the traits time needed to reach 50 % of derived from the Florida 301 9 Hicks cross. Positions of the
TG (T50) and area under the curve after 200 h of germination parents are indicated by arrows
(AUC) of untreated control seeds and seeds after CD for RILs

Florida 301 appeared to be tolerant whereas the %NG
for Hicks declined indicating that parental lines
differed in seed longevity.
Similar performance was observed when examining
the germination speed traits T50 and AUC (Fig. 2).
Control seeds of Hicks germinated faster (lower T50),
reaching higher AUC as compared to Florida 301.
After the deterioration treatment, however, the perfor-
mance of the parental lines was reversed, again
indicating the superior seed longevity of Florida 301.
The distributions of the RILs provide clear indica-
tion of quantitative inheritance of the traits under
investigation before and after the deterioration treat-
ment. The effects of the treatment were most pro-
nounced for the germination speed traits, with a
considerable increase in T50 and decrease in AUC.
QTL mapping
For the traits under consideration, seven major and two
minor QTLs on four different LGs were detected
(Table 1; Fig. 3). For the control seed lots, the highest
LOD value (6.67) was observed for a QTL for %TG
that explained 18.3 % of the variation for this trait on
LG8/18. The favorable allele at this QTL was
contributed by Hicks. One QTL affecting %NG was
detected on LG 7, along with a minor locus for %TG.
In each case the favorable allele was contributed by
Florida 301. For the control, other major and minor
QTL for T50 and AUC, respectively, were identified
in identical positions on LG 23. The low germination
speed was contributed by Florida 301 showing high
and low values for T50 and AUC, respectively.
After CD, the QTL with highest LOD (4.83) was
again detected for %TG on LG 8/18, in a similar
position as identified for the seed lots from the C. This
QTL explained 13.3 % of the phenotypic variation for
this measured trait, and the favorable allele was
contributed by Hicks. On LG 23, a QTL determining
%NG and contributed by Florida 301 was identified in
a position highly comparable to the one determining
germination speed in the control material. Finally, a
QTL controlling germination speed after treatment
appeared on LG 6 for the traits T50 and AUC. The low
speed of germination (high T50 and low AUC,
respectively) was contributed by Hicks.
Discussion
The published results on seed longevity for Nicotiana
are inconsistent. As early as 1907, Shamel and Cobey

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Table 1 Summary of the QTL mapping results of control CD), time needed to reach 50 % of total germination (T50-C,
seeds (C) and seeds after CD for the traits % total germination T50-CD) and area under the curve after 200 h of germination
(%TG-C, %TG-CD), % normal germination (%NG-C, %NG- (AUC-C, AUC-CD)
QTL Linkage group Marker Position LOD value Percent phenotypic Contributing parent
variance explained (positive allele)

%TG-C 8/18 TM10358 78.24 6.67 18.3 Hicks

T50-C 23 TM11105 0.47 3.39 12.3 Florida 301
%NG-C 7 TM10817 22.83 3.35 9.5 Florida 301
AUC-C 23 TM11105 0.47 2.63 8.1 Hicks
%TG-C 7 TM10948 19.90 2.45 7.0 Florida 301
%TG-CD 8/18 TM10142a 67.06 4.83 13.3 Hicks
%NG-CD 23 TM11105 0.47 4.80 12.9 Florida 301
T50-CD 6 TM10901 57.46 3.94 11.1 Hicks
AUC-CD 6 TM10901 57.46 3.44 9.2 Florida 301

(cited in Kingcaid 1943) stated that fully matured and
dry tobacco seed can retain viability when kept dry for
10 years, or, as has been observed in several cases, a
much longer time. Also, Johnson et al. (1930; cited in
Kingcaid 1943) reported that tobacco seed that was
originally of good germinating capacity may germi-
nate ‘satisfactorily’ at 10 years of age. More recently,
Walters et al. (2005) compared the seed longevity of
about 90 species, including N. tabacum, from different
surveys and found the germination range to decline to
50 % between 10 and 81 years. Studies by Agacka
et al. (2014) of N. tabacum and N. rustica stored at
-15/-18 °C indicate seed longevity of greater than
50 years.
Here, we describe for the first time the genetic
dissection of the seed longevity trait in N. tabacum. It
is known that germination per se and seed longevity
are very complex traits under polygenic control. In the
present study, however, only four regions, located on
four different LGs, were detected for the four traits
considered, for untreated and treated seed lots. A
major QTL for high germination percentage (TG) was
detected on LG 8/18. This seems to be a genetic factor
determining germination per se, independent of the
quality of the seed, because it was detected when both
control and deteriorated seeds were tested.
Another highly comparable region carrying a QTL,
discovered for control seeds and after the treatment,
was located on LG 23. For the untreated seed lots, this
QTL is responsible for the high germination speed of
Hicks, as shown by the presence of major and minor
QTL for T50 and AUC, respectively. After the seed
treatment, however, a QTL for high %NG of Florida
301 was found. It seems that the locus for germination
speed became negligible after the CD treatment. This
is confirmed by the phenotypic data shown in Fig. 2.
While the speed of germination for was greater for the
Hicks parent in the control seed lots, the opposite was
observed after the CD treatment. Whether this con-
tributed to the appearance of the Florida 301 QTL for
high percentage of NG in the comparable region after
the treatment remains unclear.
Two chromosomal regions, on LGs 7 and 6, were
detected separately in control and CD experiments,
respectively. On LG 7, a major and minor QTL for
%NG and %TG, respectively, were detected at highly
comparable positions. The positive alleles were con-
tributed by Florida 301. In contrast to the Hicks locus
on LG 8/18, identified in the control and CD exper-
iments, the Florida 301 QTL is expressed only with the
untreated seeds. A QTL for germination speed after
CD treatment only was detected on LG 6. The higher
speed was contributed by Florida 301which is con-
firmed by phenotypic data (Fig. 2) after CD treatment.
Considering both the phenotypic and QTL mapping
data, it can be concluded that the cultivar Hicks
expresses a high germination percentage and speed in
untreated (fresh) seeds, whereas Florida 301 seems to
withstand the controlled seed deterioration treatment
better, overtaking Hicks and, therefore, having greater
seed longevity. Thus, good germination performance
of fresh seeds does not necessarily translate into high
seed longevity. This has already been revealed for
other species. Nagel et al. (2010) investigated gene-
bank collections of barley (Hordeum vulgare), wheat
(Triticum aestivum), rye (Secale cereale), sorghum

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AUC-C

T50-C
%TG-C

% NG-C

2.63 3.39

2.45
3.35

% TG-C

6.67

7 8/18 23

%NG-CD

4.80
AUC-CD

T50-CD

%TG-CD

3.44 3.94

4.83

6 8/18 23

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b Fig. 3 Positions of the QTL of control seeds C (above) and
seeds after CD (below) for the traits % total germination (%TG-
C, %TG-CD), % normal germination (%NG-C, %NG-CD), time
needed to reach 50 % of total germination (T50-C, T50-CD),
and area under the curve after 200 h of germination (AUC-C,
AUC-CD). LODs are given below the arrows. Vertical bars
indicate intervals LOD [ 3 (major QTL; filled bars) and
LOD [ 2 (minor QTL; empty bars)
(Sorghum bicolor), oilseed rape (Brassica napus), and
flax (Linum usitatissimum) stored between 26 and
33 years. Whereas high germination was found ini-
tially, accessions separated strongly after 20 years.
Some of the ‘high germinating’ accessions dropped
close to zero.
QTL associated with seed longevity in other crops,
such as wheat and barley, often appear in genomic
regions containing genes determining aspects of biotic
and abiotic stress responses (Nagel et al. 2009, 2014;
Rehman Arif et al. 2012). The Florida 301 9 Hicks
RILs of the present study have been used for mapping
loci responsible for resistance to the black shank
disease caused by P. nicotianae (Xiao et al. 2013).
Above-ground symptoms of the disease are wilting
and yellowing of the leaves, developments that can
have a pronounced effect on seed development. One
QTL, detected in field and greenhouse disease studies
on LG 6, is comparable with the locus for germination
speed detected in the present study. The favorable
germination speed allele was contributed by Florida
301, the disease resistant parent.
The environmental growing conditions of the
mother plants have a highly significant effect on seed
quality and longevity, not only with respect to biotic
and abiotic stresses. Nagel et al. (2014) investigated
seeds of a barley association mapping population
regenerated in the same growing season but on two
different field plots separated by less than 800 m.
Beside common marker-trait associations (loci) many
‘field specific’ loci were detected. Due to the identical
regeneration year and the close proximity of the
experiments, it was concluded that soil composition
may have been one reason responsible for the differ-
ences. This was supported by a soil analysis, indicat-
ing a difference in nutrient contents, mainly
phosphorus, potassium, manganese, and nitrogen.
In order to draw final conclusions about the genetics
of seed germination/longevity and their interaction with
the growing conditions (disease resistance) of the
mother plants in Nicotiana, more studies need to be
485
performed. Another mapping population developed
from a cross between the black shank-resistant cultivar
Beinhart-1000 and Hicks (Vontimitta and Lewis 2012),
having one parent in common with the present study,
may be a good choice for follow-up investigations.
Acknowledgments We thank Sibylle Pistrick and Gabriele
Matzig for excellent technical assistance.
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