Euphytica (2011) 178:247–259
DOI 10.1007/s10681-010-0309-6
Genetic variability of seed-quality traits in gamma-induced
mutants of sunflower (Helianthus annuus L.) under
water-stressed condition
P. Haddadi • B. Yazdi-samadi • M. Berger
M. R. Naghavi • A. Calmon • A. Sarrafi
•
Received: 9 August 2010 / Accepted: 11 November 2010 / Published online: 27 November 2010
Ó Springer Science+Business Media B.V. 2010
Abstract Sunflower is one of the major annual
world crops grown for edible oil and its meal is a
potential source of protein for human consumption. It
contains tocopherol that decreases potential risk of
chronic diseases in human. The objectives of the
current research are to assess the genetic variability
and to identify AFLP markers and candidate genes
associated with seed-quality traits under well-irrigated and water-stressed conditions in gammainduced mutants of sunflower. Two mutant lines,
M8-826-2-1 and M8-39-2-1, with significant
increased level of oleic acid were identified that can
be used in breeding programs for quality increase
high oxidative stability and heart-healthy properties.
P. Haddadi
Laboratoire des Interactions Plantes Micro-organismes,
UMR CNRS-INRA 2594/441, Chemin de Borde-Rouge
Auzeville, BP 52627, 31326 Castanet Tolosan, France
The significant increased level of tocopherol in
mutant lines, M8-862-1N1 and M8-641-2-1, is justified by observed polymorphism for tocopherol pathway-related gene; MCT. The most important marker
for total tocopherol content is E33M50_16 which
explains 33.9% of phenotypic variance. One of the
most important candidate genes involving fatty acid
biosynthesis, FAD2 (FAD2-1), is linked to oleic and
linoleic acids content and explained more than 53%
of phenotypic variance. Common markers associated
with different seed-quality traits in well-irrigated and
water-stressed conditions could be used for markerassisted selection (MAS) in both conditions. Other
markers, which are specific for one condition whereas
linked to different traits or specific for a trait, could
be useful for a given water treatment.
Keywords Seed-quality traits Gamma-induced
mutants AFLP markers Candidate genes
Sunflower
P. Haddadi B. Yazdi-samadi M. R. Naghavi
Agronomy and Plant Breeding Department, Faculty
of Agriculture, University of Tehran, Karaj, Iran
P. Haddadi A. Sarrafi (&)
Laboratoire de Symbiose et Pathologie des Plantes, SP2,
IFR 40, INP-ENSAT, 18 Chemin de Borde Rouge,
BP 32607, 31326 Castanet Tolosan, France
e-mail: sarrafi@ensat.fr
M. Berger A. Calmon
UMR 1054 INRA/EIP - Laboratoire d’Agrophysiologie,
Ecole d’Ingénieurs de Purpan. 75, Voie du TOEC,
BP 57611, 31076 Toulouse Cedex 3, France
Introduction
Sunflower seed oil contains saturated and unsaturated
fatty acids, as the lipid part of the oil, as well as
tocopherol conferring antioxidant properties to the
non-lipid part of oil. Fatty acids with 18 carbons are
either saturated (C18:0; stearic acid) or unsaturated
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(C18:1; oleic acid and C18:2; linoleic acid) (Dorrell
and Vick 1997; Pérez-Vich et al. 2002). Among C18
fatty acids, oleic acid is more important because of
higher oxidative stability, more resistance to heating
and heart-healthy properties (Smith et al. 2007).
Tocopherol belongs to the Vitamin E class of lipid
soluble antioxidants that are essential for human
nutrition. The function of tocopherol in human and
animal systems is generally related to the level of
a-tocopherol activity. Alpha-tocopherol has a maximum vitamin E activity (Kamal-Eldin and Appelqvist
1996). Among oil seed crops sunflower grains mainly
contain a tocopherol, which accounts for more than
95% of the total tocopherols (Marwede et al. 2005).
In sunflower seed oil, total tocopherol content
represents the sum of a, b, c, and d tocopherol
(Ayerdi Gotor et al. 2007).
In sunflower, gamma-irradiation has been used for
inducing genetic variability for different characters
such as osmotic-related traits (Poormohammad Kiani
2007), resistance to Phoma macdonaldii (Abou Al
Fadil et al. 2004), germination traits (Alejo-James
et al. 2004), morphological traits (Nabipour et al.
2004) and organogenesis (Rachid Al-Chaarani et al.
2004). Mutagenesis has been successfully used for
developing variation in the fatty acid profile of
sunflower and some mutants with altered fatty acid
content have been developed (Osorio et al. 1995;
Fernández-Martı́nez et al. 1997; Cantisán et al. 2000;
Lacombe and Berville 2001; Pérez-Vich et al. 2004;
Schuppert et al. 2006). High palmitic acid mutants;
275HP, CAS-5 and CAS-12 (Fernández-Martı́nez
et al. 1997), and the high stearic acid line, CAS-3, as
well as two lines with midstearic acid content, CAS-4
and CAS-8, were obtained (Osorio et al. 1995).
Developing midstearic acid sunflower lines (CAS-19,
es1es1Es2Es2, and CAS-20, Es1Es1es2es2) from a
high stearic acid mutant is also reported (Pérez-Vich
et al. 2004). The genetic studies of CAS-3, CAS-4
and CAS-8 revealed that total stearic acid increased
as a result of reduced conversion rate of stearic to
oleic acid while conversion rate of palmitic to stearic
was not changed (Cantisán et al. 2000). Enzymatic
actives for stearoyl-ACP desaturase and acyl-ACP
thioesterase in above-mentioned high stearic acid
mutant showed that stearoyl-ACP desaturase activity
was reduced whereas acyl-ACP thioesterase activity
was increased (Cantisán et al. 2000). Prevenets, high
oleic acid mutants, have been so far developed by
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Euphytica (2011) 178:247–259
chemical mutagenesis (dimethyl-sulfate) (Soldatov
1976). In genotypes with high oleic acid content, in
addition to 5.7 kb an extra 7.9 kb EcoRI restriction
fragment (EcoRI-D12HOS) was observed in comparison with genotypes with normal oleic acid content. A
novel HindIII restriction fragment of more than 15 kb
(HindIII-D12HOS) instead of 8 kb was also reported
(Lacombe and Berville 2001). Co-segregation of
FAD2-1 with Ol in high-oleic sunflower mutant was
also reported (Schuppert et al. 2006). Three loci,
Tph1 (m), Tph2 (g) and d, can control the level of a
tocopherol in sunflower seed (Hass et al. 2006; Tang
et al. 2006; Vera-Ruiz et al. 2006). The amount of b
tocopherol is increased by d locus in mutant inbred
lines (m m) whereas the level of c tocopherol is
enhanced by g locus in mutant inbred lines (g g) as a
result of knockout of c tocopherol methyl transferase
(Hass et al. 2006). 2-Methyl-6-phytyl-1,4-benzoquinone/2-methyl-6-solanyl-1,4-benzoquinone methyltransferase (MPBQ/MSBQ-MT) paralogs from
sunflower (MT1 and MT2) were isolated and
sequenced (Tang et al. 2006). INDEL markers were
developed for MT1 and MT2 and the MT1 Locus was
mapped to linkage group 1 (Tang et al. 2006). In this
research the genetic variation of seed-quality traits
such as total tocopherol, protein, oil and fatty acids
contents, as well as polymorphism for AFLP markers
and some candidate genes (CGs) in gamma-induced
mutants of sunflower under well-irrigated and waterstressed conditions are studied.
Materials and methods
Plant materials and experimental conditions
The sunflower restorer inbred line ‘AS613’ has
been produced in our laboratory from a cross between
two genotypes (‘ENSAT-125’ and ‘ENSAT-704’)
through a single-seed descent (SSD) programme
(Sarrafi et al. 2000). The seeds of ‘AS613’ were
exposed to gamma rays at the Atomic Energy Center
(Cadarache, France) with a dose of 75 Grays. Mutants
population have been developed through modified
SSD method (Sarrafi et al. 2000). Regarding to
morpho-physiological studies, among a population of
about 2000 gamma-induced mutants of sunflower, 23
M8 mutants were selected for quantitative analysis.
Two experiments were undertaken in randomized
Euphytica (2011) 178:247–259
complete block design (water treatment as main plot)
with three replications at Tehran University-Iran2007. Seeds of mutants and original line (AS613)
were sown in the field under well-irrigated and waterstressed conditions. Each genotype per replication
consisted of one row, 4 m long, 50 cm between rows
and 25 cm between plants in rows. The distance
between replications of well-irrigated and waterstressed treatments was 7 m. The so-called ‘wellirrigated’ condition plots were irrigated once a week,
whereas for the second condition (water-stressed),
water deficit was started 45 days after sowing at the
stage near flower bud formation and continued up to
maturity.
249
Solvent extraction of lipids
The extraction of the total oil content was performed
by hexane (n-hexane, Prolabo/Subra, Toulouse,
France) extraction using an accelerated solvent
extractor apparatus (ASE 200, Dionex, France) with
an isopropanol/hexane mixture (5:95 v/v) during
20 min. Then, the solvent was removed from the
extracts under low-pressure evaporation (Rotavapor,
Bioblock Scientific HS 40 HUBER, Heildorph,
Germany). Lipid extracts were weighed and tocopherol content was analyzed.
Tocopherol determination
Trait measurements
Seed-quality traits
Near infrared reflectance (NIR) spectroscopy, has
been successfully used as an alternative technique to
classical methods to determine multiple parameters
of seed quality traits in sunflower, such as proteins,
oil content, fatty acid compositions (Pérez-Vich et al.
1998; Velasco and Becker 1998; Biskupek-Korell
and Moschner, 2007; Ebrahimi et al. 2008, 2009).
Seed protein content (SPC), seed oil content (SOC),
palmitic acid content (PAC), stearic acid content
(SAC), oleic acid content (OAC) and linoleic acid
content (LAC) were measured in mutants and
original line (AS613) in each replication for both
conditions by the FOSS NIRSystems 6500. Twenty
grams of sunflower seeds per genotype per condition
per replication were ground in a Knifetec 1095
Sample Mill (1975, Foss Tecator, Höganäs, Sweden)
three times for 10 s each. A FOSS NIR Systems
6500 spectrophotometer (Foss Analytical, Denmark)
was used to collect spectra from the ground
sunflower seeds using a small round cup with a
quartz window. The reflectance (R) of each sample
was measured as log of 1/R from 400 to 2500 nm at
2 nm intervals.
Pre-measurements for total tocopherol content
(TTC) were carried out by both FOSS NIRSystems
6500 and reference method (HPLC, ISO 9936, 1997)
for core collection (44 samples). Total oil content was
extracted and TTC was thus determined using the
following subsections.
Total tocopherol was achieved using a high-performance liquid chromatography (HPLC) (SpectraPhysics, Thermo Separation Products, USA) with a
normal-phase LiChrosorb Si60 column, 250 cm 9
4 mm 9 5 lm (CIL, Cluzeau, France) (ISO 9936,
1997). The mobile phase was a mixture of hexane/
isopropanol (99.7:0.3 v/v) at 1 ml/min flow rate. One
gram of oil sample was diluted in 25 ml of hexane
and 20 ll was injected into the HPLC. Detection
was performed with fluorescence detector (excitation wavelength = 298 nm and emission wavelength = 344 nm: Waters 2475 multi k). Total
tocopherol content was calculated as the sum of a,
b, c, and d-tocopherol contents and expressed in
mg kg-1 oil. A modified partial least-squares regression (MPLS) model, after 4 outlier elimination passes
(WINISI 1.02—Infrasoft International LLC) was
used. The performance of our NIRS model, for the
estimation of tocopherols was determined by the
following parameters: the standard error of calibration (SEC), the coefficient of determination in
calibration (RSQ), the standard error of cross-validation (SECV), the coefficient of determination of
cross-validation (1 - VR) and the standard error of
prediction (SEP).
We have obtained a high significant correlation
between the HPLC analysis and the NIRS
predictions for TTC (R2 = 0.76) indicating the
NIRS method can be used to determine total
tocopherol content. Then, TTC was measured in
mutants and original line (AS613) in each replication for both conditions by the FOSS NIRSystems 6500.
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Euphytica (2011) 178:247–259
Molecular analysis
The genomic DNA of original and mutant lines were
isolated according to the method of extraction and
purification presented by Porebski et al. (1997) and
DNA quantification was performed by picogreen. The
AFLP procedure is previously described by Darvishzadeh et al. (2008). Different MseI/EcoRI primer
combinations were used for AFLP genotyping
(Table 1). Polymorphism of some important candidate genes; tocopherol pathway-related, phosphoglyceride transfer-related, enzymatic antioxidant-related,
drought-responsive and fatty acid biosynthesis-related
genes were studied. Reactions catalyzed by proteins of
the tocopherol and fatty acid pathways are illustrated
in Figs. 1 and 2, respectively. Respective sequence
data for candidate genes coding for these proteins
were obtained from The Arabidopsis Information
Resource, TAIR, (www.arabidopsis.org). In order to
seek the helianthus homolog sequences to the Arabidopsis genes, we used the Compositae EST assembly clusters, available at the Helianthus-devoted
bioinformatics portal Heliagene (www.heliagene.org).
Table 1 Seventeen AFLP primer combinations and their
polymorphic markers used for genotyping sunflower mutants
and their original line (AS613)
Primer combinations
Number of
polymorphic
markers
E40M59(EAGC 9 MCTA)
9
E40E50(EAGC 9 MCAT)
E38M62(EACT 9 MCTT)
9
8
E37M50(EACG 9 MCAT)
27
E37M62(EACG 9 MCTT)
9
E37M48(EACG 9 MCAC)
8
E33M50(EAAG 9 MCAT)
16
E33M49(EAAG 9 MCAG)
15
E33M59(EAAG 9 MCTA)
15
E33M47(EAAG 9 MCAA)
13
E33M60(EAAG 9 MCTC)
11
E33M62(EAAG 9 MCTT)
10
E33M48(EAAG 9 MCAC)
9
E33M61(EAAG 9 MCTG)
8
E31M50(EAAA 9 MCAT)
13
E31M62(EAAA 9 MCTT)
10
E31M48(EAAA 9 MCAC)
Total
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10
200
The Helianthus EST clusters presenting the reciprocal
blast with the highest score and lowest E value with
regarding to the original Arabidopsis genes were chosen for our studies. All Primers were designed by
MATLAB. Four various primer combinations per each
candidate gene were tested on agarose gel. Primers used
for PCR are summarized in Table 2. The PCR program
was: 4 min at 94°C followed by 35 cycles; 30 s at 94°C,
30 s at 55°C, 1 min at 72°C and at last, 5 min at 72°C.
Statistical analysis
The data were analyzed using SPSS. The association
between AFLP markers and candidate genes with the
quantitative traits was estimated through stepwise
multiple regression analysis, where each quantitative
trait was considered as a dependent variable while
AFLP markers and candidate genes were treated as an
independent variable. To select independent variables
for the regression equation, F-values with 0.045 and
0.099 probabilities were used to enter and remove,
respectively. Multiple regression analysis has been
used to identify molecular markers associated with
morphological and yield traits in some crops (Virk
et al. 1996; Vijayan et al. 2006).
Results
Phenotypic variation
Results of analysis of variance show significant
genotypic effect (Mutants) for seed-quality (Table 3)
trait under well-irrigated and water-stressed conditions. The effect of water treatment is not significant
for unsaturated fatty acid content (oleic and linoleic
acid) whereas it is significant for TTC, SPC, SOC and
saturated fatty acid content (Table 3). Characteristics
of sunflower M8 mutant lines in both conditions for
seed-quality traits are also summarized in Table 3.
Regarding the range of mutant lines, variation for all
studied traits was observed and some mutants
presented significant higher values compared with
the original line.
Molecular analysis
Seventeen AFLP primer combinations and their
polymorphic markers used for genotyping mutants
2-C-methyl-D-erythritol 4-phosphate
Arogenic acid
MCT
Tyrosine
AS613
Shikimate pathway
M8-641-2-1
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M8-862-1N1
Euphytica (2011) 178:247–259
1600 bp
1200 bp
2-C-methyl-D-erythritol 4 (Cytidine 5’-phospho)
p-hydroxyphenylpyruvate
Isoprenoid
Homogentisic acid
Phytyl pyrophospate
Homogenitisate phytyltransferase
(VTE2)
-tocopherol
VTE4
-tocopherol
M8-381-1-1
2, 3-Dimethyl-5-phytyl-1, 4- benzoquinol
M8-641-2-1
2-Methyl-6-phytyl-1,4-benzoquinol (MPBQ)
2000 bp
-tocopherol
-tocopherol methyl-transferase
(VTE4)
-tocopherol
Fig. 1 Tocopherol biosynthetic pathway; ‘2-C-methyl-D-erythritol 4-phosphate’ is converted into ‘2-C-methyl-D-erythritol 4
(Cytidine 50 -phospho)’ by 2-C-methyl-D-erythritol 4-phosphate
cytidyl transferase (MCT).‘2-Methyl-6-phytyl-1,4-benzoquinol’ (MPBQ) is formed after the condensation of homogentisic
acid (HGA) and phytyl pyrophosphate (PDP) by homogenitisate phytyltransferase (VTE2). a-tocopherol can be generated
by methylation of c-tocopherol via c-tocopherol methyl-
transferase (VTE4) (D’Harlingue and Camara 1985).
b-tocopherol is formed from d-tocopherol by methylation of
the 5 position by VTE4 (Norris et al. 2004).The studied
candidate genes are also highlighted in bold. Gel electrophoretic separation of candidate gene–PCR products from original
line (AS613) and some mutants are presented with the
corresponding metabolic pathway
and their original line (AS613). The number of
polymorphic markers varied from 8 to 27 for different
primer combinations (Darvishzadeh et al. 2008).
Polymorphisms are also observed for the studied
candidate genes for original line (AS 613) and some
mutants (Fig. 3). The results of marker identification
for different traits under well-irrigated and waterstressed conditions are summarized in Table 4.
Results revealed that the number of AFLP marker
and candidate gene (CG) associated with seed-quality
traits ranged from 4 to 6 depending on trait and
conditions. The percentage of phenotypic variance
(R2) explained by each marker or candidate gene
associated with the traits ranged from 4.4% to 53.3%.
The most important marker for TTC is E33M50_16
and explained 33.9% of phenotypic variance. One of
the most important candidate genes involving tocopherol pathway (Fig. 1), homogenitisate phytyltransferase (VTE2), is linked to TTC (Table 4). The
E31M50_12 marker correlated with SPC is identified in well-irrigated condition and explained 35%
of phenotypic variance. The largest amount of
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Euphytica (2011) 178:247–259
FACII
AS 613
M8-52-1-1
Palmitic acid (16:0)
2000 bp
Stearoyl ACP desaturase – PC 19
500 bp
Triacylglycerols
M8-826-2-1
stearoyl-ACP
desaturase
Oleic acid (18:1)
M8-39-2-1
FatB- PC 22
acyl-ACP
thioesterase
AS 613
Stearic acid (18:0)
1500 bp
FAD2-1– PC 18
900 bp
FAD2
FAD2-2 – PC 17
500 bp
FAD2-3 – PC 14
linoleic acid (18:2)
FAD2-3 – PC 15
600 bp
Fig. 2 Simplified fatty acid biosynthetic pathway. Stearic acid
is formed from palmitic acid by FACII, which lengthens
palmitic acid (16:0) by two carbon atoms to produce stearic
acid (18:0) (Pleite et al. 2006). Stearic acid can be either
desaturated by D9-desaturase (stearoyl-ACP desaturase) which
catalyses the first desaturation of stearic acid (18:0) to oleic
acid (18:1) or hydrolyzed by acyl-ACP thioesterase (Heppard
et al. 1996; Lacombe and Berville 2001; Vega et al. 2004).
Finally, linoleic acid is formed from oleic acid by D12desaturase (oleoyl-PC desaturase; FAD2), which catalyses the
second desaturation of oleic acid (18:1) to linoleic acid (18:2)
(Garcés and Mancha 1991). The studied candidate genes are
also highlighted in bold. Gelelectrophoretic separation of
candidate gene–PCR products from original line and mutants
are presented with the corresponding metabolic pathway. PC
primer combination
phenotypic variance (R2) explained by E37M50_10
for SOC is 35.3%. Under water-stressed condition,
association between SEC14 and SOC is observed.
Among 11 identified markers for PAC in both
conditions, E37M50_20 is most important with
R2 = 28.6%. Under well-irrigated condition, the
correlation between POD and PAC is also observed.
Two common markers; E33M59_6 and E31M50_5,
are detected for SAC. One of the most important
candidate genes involving fatty acid biosynthesis
(Fig. 2), FAD2 (FAD2-1), is linked to OAC and LAC
and explains more than 53% of phenotypic variance
(Table 4).
traits. Mutant line, M8-862-1N1, presents significant
increased level of tocopherol (403.78 mg kg-1oil
compared with 314.3 mg kg-1oil in original line AS613; Table 3). Mutant lines, M8-826-2-1 and M8-392-1, with significant increased level of oleic acid
(70.3 mg 100 mg -1oil in M8-826-2-1 mutant compared with 29.2 mg 100 mg-1oil in original line AS613; Table 3) are developed by gamma rays with a
dose of 75 Grays in our research. These mutants can be
used in breeding programs because of high oxidative
stability and heart-healthy properties.
Molecular genetic studies have been carried out in
the aforementioned population through AFLP markers and candidate genes (CGs). The results of marker
identification show that some AFLP markers and
candidate genes are associated with several traits and
some others are specific for only one trait (Table 4).
Among all studied candidate genes involving tocopherol biosynthetic pathway, polymorphisms were
observed for VTE4, VTE2 and MCT genes among
some mutants and original line (Fig. 1). Endrigkeit
et al. (2009) reported that in rape seed genotypes,
Discussion
The large genetic variability observed among mutant
lines for the studied traits revealed that the efficiency of
gamma-irradiation for inducing genetic variation in
sunflower for seed-quality traits. Some mutants have
advantages over the original line ‘AS613’ for different
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Target gene
Accession
AGI-Arabidobsis
Homologue with
Heliagene cluster
Primer pair
combination
Sequence of primer (50 –30 )
Forward
Reverse
Tocopherol pathway-related genes
VTE4
AT1G64970.1
HuCL02246C001
1
ATCCGTATGATTGAACAAGC
ATGTGCTCTCCACTCTCCATTG
2
GTTTGGTCAATGGAGAGTG
ATCCTTCAATCATTAGTGGC
VTE2
AT2G18950.1
HuCL02840C003
3
TGCCACAAGAGCAAATCGCTTC
TTTGGGCACTCTTCATAAG
MCT
AT2G02500.1
HuCL00002C009
4
CAAAGTCTTCACCACAAATG
ACCTCATCCCATCTTCTTCC
Euphytica (2011) 178:247–259
Table 2 Primers used for PCR
Phosphoglyceride transfer-related genes
PGT
Cytosolic
AT1G75170.1
AT3G24840.1
HuCL10527C001
HuCL09897C001
5
6
TATGTCCATCTTTCGGCGTC
ATGATAACCGTGTGGATAGC
ATGGTGTCTTTAGCGGTTC
ATGCTAAACTGGAGGAAAGC
SEC14
AT2G21540.2
HuCL00667C001
7
CAAGGAAGGATTTCACCGTG
AAGGCGGTTGATGCTTTACG
Enzymatic antioxidant-related genes
POD
AT1G14540.1
HuCL03143C001
8
GACTTGGAAGAAGAGATTCAC
ATTGTCAGCATACTCGGTC
CAT
AT1G20620.1
HuCL00001C054
9
AAACTACCCTGAGTGGAAG
AATGAATCGTTCTTGCCTG
GST
AT1G02930.1
HuCL00790C003
10
AAAGAGCACAAGAGTCCTG
ACTTATTTGAGTGGGCAAC
Drou
AT5G26990.1
HuCL02051C001
11
TTGTTGAGGAGGGAACTAAG
GTCATCACCAAGAATCGTCG
SPL2
AT5G43270.1
HuCL10252C001
12
ATTTGATGGGAAGAAGCGG
CATTGTGGTCAGAAAGCCTC
Dehydrin
AT3G50980.1
HuCL00053C009
13
AAGTTCTCCAAACCGACGAG
ACAACCACAGTGAAACCAC
Drought-responsive genes
Fatty acid biosynthesis-related genes
FAD2a
FAD2-3
_
AY802998.1
14
GCCTTATTCTACATTCTGCTC
ATCCCATAGTCTCGGTCTAC
HuCL07925C001
15
GCCTTATTCTACATTCTGCTC
AATCGCCTTTGTTGCTTCC
FAD2-2
_
AY802993.1
16
GGTCTGTCATCCGTTCATTC
GCGAATCGGTCATAATACC
FAD2-1
_
HuCL00141C001
DQ075691.1
17
18
GGTCTGTCATCCGTTCATTC
GAGAAGAGGGAGGTGTGAAG
AGTCCCGTCAAACTGATAG
GCCATAGCAACACGATAAAG
HuCL00406C001
19b
GAGAAGAGGGAGGTGTGAAG
ACAAAGCCCACAGTGTCGTC
HuCL00103C001
20
GACGTTTCAATCAGACCTGT
GCATTGTTTGGTAAGTAGGC
21
GCCTACTTACCAAACAATGC
TATTTTTGTGTAGGCGGTTT
Stearoyl ACP desaturase
AT1G08510.1
HuCL03123C002
22
TTACACATTCGGCTTATCG
TGGTTGATAAAGGTTCTCGGG
23
TTACACATTCGGCTTATCG
GCACATTTCTGGTGTTGAACCG
24
ACTGAGGTGAATGGGAGTAG
GCACATTTCTGGTGTTGAACCG
253
123
FatB
AT2G43710.1
254
123
Table 2 continued
Target gene
Accession
FatA
AGI-Arabidobsis
Homologue with
Heliagene cluster
AT3G25110.1
HuCL04107C001
Primer pair
combination
Sequence of primer (50 –30 )
Forward
Reverse
25
AATAAGACGGCGACTGTTG
TTCAATTTCAACCACATCA
The candidate genes are: tocopherol methyl-transferase (VTE4), homogenitisate phytyltransferase (VTE2), 2-C-methyl-D-erythritol 4-phosphate cytidyl transferase (MCT),
phosphoglyceride transfer (PGT), cytosolic, phosphatidylinositol transporter (SEC14), peroxidase (POD), glutation-s-transferase (GST), catalase (CAT), drought-responsive
(Drou, SPL2), dehydrin, FAD2 (FAD2-1, FAD2-2, FAD2-3), stearoyl ACP desaturase and acylACP thioesterase (FatA, FatB)
a
Sequences of FAD2 used for primer design were obtained from GenBank
b
Specific primer for high OAC*(Bervillé et al. 2009)
Table 3 Characteristics of sunflower M8 mutant lines for seed-quality traits in well-irrigated (WI) and Water-stressed (WS) conditions
Trait
Mutants
WI
Mean
314.3
26.8
32.6
5.9
5.8
29.2
59.5
WS
277.5
27.2
32.0
6.5
7.0
28.1
60.6
Some mutant lines
Range
Effect
M8-826-2-1
M8-133-2
M8-186-1
M8-143-2
M8-862-1N1
Ma
Wb
WI
WS
WI
WS
WI
WS
WI
WS
WI
WS
WI
WS
WI
WS
WI WS
312.1
27.26
34.8
5.9
6.0
29.1
60.2
274.9
27.9
32.4
6.0
6.8
29.7
59.6
228.9–403.7
22.2–31.0
27.2–42.5
4.6–7.1
5.1–7.4
13.2–65.3
26.0–75.9
134.9–395.4
23.2 –31.1
27.0–39.7
4.8–6.8
5.1–7.3
14.3–68.8
23.0–74.6
292.9
30.0
36.3
4.7*
5.8
70.3*
26.0*
303.1
31.2
35.9
4.8*
6.3
68.8*
23.0*
284
31.0*
27.8
5.5
6.7
29.1
59.3
261.5
30.0*
29.8
6.1
7.3
22.9
65.7
387.4*
22.2
42.5*
7.0**
5.3
13.2*
75.9**
365.1*
27.7
33.8
6.8
6.1
14.3*
74.5**
342.1
30.5
37.3*
6.4
7.4*
35.2
53.9
345.7
30.0
35.7*
6.1
6.8
36.6
52.6
403.7*
24.4
40.7*
6.5
5.2
18.9
70.6
395.5*
23.3
39.2*
6.9
5.2
16.3
72.9
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
*
**
*
**
NS
NS
All significant increasing or decreasing are highlighted in bold
Note: TTC total tocopherol content, SPC seed protein content, SOC seed oil content, PAC palmitic acid content, SAC stearic acid content, OAC oleic acid content, LAC linoleic acid content
*, ** Significant difference with original line (AS613) at 0.05 and 0.01 probability level, respectively
a
M mutants effect
b
W water treatment effect
Euphytica (2011) 178:247–259
TTC (mg kg-1 oil)
SPC (g 100 g-1 dry matter)
SOC (g 100 g-1 dry matter)
PAC (mg 100 mg-1 oil)
SAC (mg 100 mg-1 oil)
OAC (mg 100 mg-1 oil)
LAC (mg 100 mg-1 oil)
Original line
(AS613)
Euphytica (2011) 178:247–259
255
Table 4 AFLP markers and candidate genes correlated with seed-quality traits in a population of sunflower mutants and original line
(AS613) under well-irrigated and water-stressed conditions
Traits
TTC
SPC
SOC
PAC
Well-irrigated condition
OAC
LAC
Water-stressed condition
P-Value
Marker
R2%
6.8
<0.0001
E40M59_5
30.7
6.7
<0.0001
10.1
\0.0001
E37M50_7
15.7
5.1
<0.0001
5.5
<0.0001
E33M59_7
11.2
3.5
0.003
7.0
5.1
\0.0001
E33M59_2
10.7
3.5
0.002
E37M50_7
5.0
3.0
0.007
7.0
2.6
0.01
E31M50_12
35.0
8.4
\0.0001
E37M50_12
23.0
7.7
\0.0001
E37M50_10
19.0
5.8
<0.0001
E40M59_7
19.2
4.8
<0.0001
E31M48_1
11.2
5.2
\0.0001
E33M61_6
13.8
6.2
<0.0001
E37M62_7
12.1
5.2
\0.0001
E33M49_7
16.0
7.2
\0.0001
E40M59_7
6.1
3.8
<0.0001
E33M47_2
11.3
3.5
0.003
E37M50_10
E31M50_6
35.3
22
7.1
6.3
<0.0001
<0.0001
E33M59_4
E33M47_6
21.3
32.0
6.4
7.7
\0.0001
\0.0001
E38M62_7
12.9
7.3
<0.0001
E37M50_6
14.3
7.4
<0.0001
E38M62_8
10.5
4.4
\0.0001
E33M47_1
10.4
4.9
\0.0001
Marker
R%
E37M50_9
32.7
E33M50_16
33.9
E40M59_5
10.5
E40M50_7
T- Value
VTE2
T- Value
P-Value
E37M50_10
9.1
4.1
0.001
SEC14
4.9
3.2
0.005
E33M60_8
26.8
6.3
<0.0001
E37M50_20
28.6
10.1
<0.0001
FAD2-1
25.1
6.8
<0.0001
E33M61_5
22.9
7.9
\0.0001
E40M59_6
13.6
3.6
0.002
E40M50_8
17.0
6.0
\0.0001
E33M50_13
6.9
3.5
0.003
E37M50_3
8.5
6.1
\0.0001
E33M59_11
6.0
3.2
0.005
E33M50_8
8.7
3.9
0.001
6.7
2.7
0.01
E33M50_13
28.9
9.6
\0.0001
E33M61_3
37.4
5.5
\0.0001
E33M59_6
27.3
8.8
<0.0001
E33M59_6
21.7
6.0
<0.0001
E31M50_5
14.1
5.2
<0.0001
E31M50_5
14.1
6.0
<0.0001
Drou
E33M61_6
12.1
7.5
5.3
3.7
\0.0001
0.002
E37M50_20
E33M60_4
10.2
6.3
4.0
3.3
0.001
0.004
<0.0001
POD
SAC
2
FAD2-1
44.3
11.7
\0.0001
FAD2-1
52.8
7.5
E33M60_8
19.4
3.6
0.002
E33M60_8
16.4
3.9
0.001
E33M59_10
8.0
5.4
\0.0001
E33M61_1
7.5
2.8
0.012
E33M59_8
9.0
4.3
\0.0001
E37M50_9
6.2
2.6
0.017
E33M59_11
4.4
3
<0.0001
0.009
FAD2-1
43.7
9.5
\0.0001
FAD2-1
53.3
9.7
E33M60_8
19.3
6.1
<0.0001
E33M60_8
18.2
5.7
<0.0001
E40M59_6
8.6
3.3
0.004
E38M62_6
6.8
4.4
\0.0001
E33M49_13
6.6
4.2
0.001
E33M47_10
7.1
3.9
0.001
TTC total tocopherol content, SPC seed protein content, SOC seed oil content, PAC palmitic acid content, SAC stearic acid content.
The candidate genes are: homogenitisate phytyltransferase (VTE2), phosphatidylinositol transporter (SEC14), peroxidase (POD),
drought-responsive (Drou). Common markers are also shown as bold face
VTE4 was anchored to the end of chromosome A02,
where also two QTLs for a-tocopherol content had
been identified. Primer combination corresponding to
MCT gene (PC 4; Table 2) led to PCR fragment of
about 1.2 kb in M8-862-1N1 and M8-641-2-1 Lines
and 1.6 kb in AS613 (Fig. 1). The increased level of
123
M8-78-1
M8-653
M8-133-2
M8-352-2-2
M8-381-1-1
AS 613
M8-263-2
M8-186-1
M8-135-2
M8-36-2
M8-338-2-2
M8-16-2
M8-39-2-1
M8-641-2-1
M8-862-1N1
M8-575-1
M8-652-1
M8-826-2-1
M8-143-2
M8-32-1-1
M8--133-1
M8-375-1
M8-52-1-1
Fig. 3 Polymorphisms for
candidate gene–PCR
products among original
line (AS613) and mutants.
a phosphatidylinositol
transporter (SEC14)-PC7,
b catalase (CAT)-PC9,
c dehydrin-PC13,
d peroxidase (POD)-PC8
and e phosphoglyceride
transfer (PGT)-PC5. PC
Primer combination
Euphytica (2011) 178:247–259
M8-417-1
256
2500 bp
2000 bp
1500 bp
a
1000 bp
800 bp
b
1000 bp
c
1500 bp
1000 bp
800 bp
d
2500 bp
2000 bp
1500 bp
e
tocopherol content in both lines with a truncated
MCT gene might be explained by the possible
deletion of an inhibitor-binding domain in the
promoter region of the gene. The polymorphisms
for some Phosphoglyceride transfer-related genes are
also observed among mutants and original line
(Fig. 3).
The level of TTC, SOC, PAC and LAC are
significantly increased in M8-186-1 line (Table 3)
and polymorphism for phosphatidylinositol transporter (SEC14) is also observed compared with
AS613 (Fig. 3). The level of SOC is significantly
increased in M8-52-1-1 line (40.2 g 100 g-1 dry
matter in M8-52-1-1 mutant compared with 32.6 g
100 g-1 dry matter in original line AS-613) and
polymorphism for stearoyl ACP desaturase, phosphatidylinositol transporter (SEC14), CAT, POD and
PGT genes is also observed compared with AS613
(Figs. 2, 3). It has been reported that SEC14 domains
exist in proteins from plants, yeast and mammals
(Saito et al. 2007). Wide range of lipids, phosphatidylglycerol and tocopherols were known as ligands
for SEC14 domain-containing proteins (Saito et al.
2007).
Some enzymatic antioxidant-related genes such as
peroxidase (POD) and catalase (CAT) present polymorphisms in some mutant lines compared with the
123
original line; AS613 (Fig. 3). The association
between POD gene, enzymatic antioxidant, and
PAC is observed (Table 4). The interdependence
between antioxidant and lipid peroxidation has been
reported (Semchuk et al. 2009). In plants, the
protection of photosynthetic apparatus and polyunsaturated fatty acids from oxidative damage caused
by reactive oxygen species (ROS) are the main
function of antioxidant (Trebst et al. 2002; Cela et al.
2009; Semchuk et al. 2009). Primer combination
corresponding to Dehydrin gene (PC 13; Table 2) led
to specific PCR fragment of about 1 kb in M8-417-1,
M8-826-2-1, M8-39-2-1 and M8-186-1 Lines
(Fig. 3). Dehydrin is a gene of the D-11 subgroup
of late-embryogenesis-abundant (LEA) proteins
(Dure et al. 1989; Close et al. 1993), associated with
drought tolerance in sunflower (Ouvrard et al. 1996;
Cellier et al. 1998). In sunflower three FAD2 genes
have been isolated whereas from both cotton and
soybean two and from Arabidopsis only a single
FAD2 genes have been identified (Heppard et al.
1996; Liu et al. 1997; Martı́nez-Rivas et al. 2001). In
the present study, sequences of FAD2 used for primer
design were obtained from GenBank, closet homologue in Helianthus is indicated as reference. Primer
combinations corresponding to FAD2_1 gene (PC 18;
Table 2) led to high oleic acid specific amplification
Euphytica (2011) 178:247–259
fragments of about 1.5 kb in M8-826-2-1 and M8-392-1 Lines. In contrast, primer combinations corresponding to FAD2_2 and FAD2_3 genes just led to
nonspecific bands in mutants and original line
(Fig. 2). These results suggest that the high oleic
mutation in gamma-induced sunflower population
interferes with the mutation in FAD2_1 gene. FAD21 is seed specific and strongly expressed in developing seeds (Martı́nez-Rivas et al. 2001). Co-segregation of FAD2-1 with Ol gene has been shown and it
has been also assigned to linkage group 14 in
sunflower (Schuppert et al. 2006). The high association between FAD2-1 gene and E33M60_8 with
OAC and LAC can be explained by correlation
between OA and LA as well as by a specific gene for
D12-desaturase (oleoyl-PC desaturase), which catalyses the second desaturation of oleic acid (18:1) to
linoleic acid (18:2) (Garcés and Mancha, 1991). High
oleic acid mutants can be developed either by the
upstream desaturation of stearic acid into oleic acid
by D9 desaturase or by the downstream desaturation
of oleic acid by D12 desaturase. High significant and
negative correlation between OA and LA (Ebrahimi
et al. 2008) is justified by opposite coefficient of their
common markers (Table 4). This phenomenon poses
potential challenges to breeders for simultaneous
improvement of both traits. However, independent
markers for OA and LA identified in our research
(Table 4) provide opportunity for simultaneous
improvement of these two traits in sunflower.
Two stable markers, E40M59_5 and E37M50_7,
for TTC are identified in both conditions (Table 4).
The changes in TTC during plant responses to drought
stress can be characterized by two phases. In the first
phase, increased TTC contribute to avoid oxidative
damage by quenching reactive oxygen species (ROS).
The second phase occurs when the stress is severe.
TTC decreases during the second phase and consequently, lipid peroxidation increases and cell death
happens if tocopherol deficiency cannot be compensated by other mechanisms of protection (MunnéBosch 2005). An increase of tocopherol synthesis
under moderate stress and a decrease of tocopherol
synthesis under severe stress have been reported
(Munné-Bosch 2005). Under water-stressed condition, common marker; E37M50_20, for PAC and
SAC is detected (Table 4). This can be explained by a
specific gene for fatty acid synthetase II (FACII),
which lengthens palmitic acid (16:0) by two carbon
257
atoms to produce stearic acid (18:0) (Pleite et al.
2006). Common markers associated with different
seed-quality traits in well-irrigated and water-stressed
conditions could be used for marker-assisted selection
(MAS) in both conditions. Other markers, which are
specific for one condition whereas linked to different
traits or specific for a trait, could be useful for a given
water treatment.
Acknowledgments This research was supported by
Gundishapur project No. 12267RD. The authors wish to
thank the French and Iranian governments for providing
fellowship for Ph.D. program of the first author.
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