Identification and Characterization of Genes Conferring Reduced Saturate Oil in
Sunflower (Helianthus annuus L.)
James Gerdes1, Wenxiang Gao2, Robert Benson3, Angela Erickson4, and Charles Kahl5.
1
Dow AgroSciences, 2230 Hwy 75 North, Breckenridge, MN 56520, USA, jtgerdes@dow.com,
2
Dow AgroSciences, 9330 Zionsville Rd, Indianapolis, IN 46268, USA, wgao2@dow.com,
3
Dow AgroSciences, 2230 Hwy 75 North, Breckenridge, MN 56520, USA, rmbenson@dow.com,
4
Dow AgroSciences, 2230 Hwy 75 North, Breckenridge, MN 56520, USA, alerickson@dow.com,
5
Dow AgroSciences, 9330 Zionsville Rd, Indianapolis, IN 46268, USA, cjkahl@dow.com.
ABSTRACT





The reduction of saturated fatty acids in sunflower would greatly improve the quality resulting in
healthier oil. The objectives of this research were to identify and characterize quantitative trait
loci (QTL) that reduce the primary saturated fatty acids in sunflower, palmitic acid (C16:0) and
stearic acid (C18:0).
Accumulated gas chromatography (GC) data from diverse Dow AgroSciences (DAS) proprietary
germplasm was methodically reviewed resulting in the identification of naturally occurring
variants for both reduced C16:0 (lpm) and reduced C18:0 (lst). These variants were
independently crossed to develop F2 mapping populations. The variants were also crossed
together to evaluate combined effects in total saturate reduction.
Major recessive QTL were identified for both lpm and lst using SSR markers. A QTL on LG5
explained 71.9% of the phenotypic variation for C16:0 and a QTL on LG17 explained 36.1% of
the phenotypic variation for C18:0. Linked markers were identified and are being used to
introgress these traits into elite inbred lines. Progeny from crosses between lpm and lst in a high
oleic (Fad2-1) genetic background demonstrated total saturate levels as low as 2.33%.
The total saturated fatty acid content of sunflower oil can be greatly reduced using the QTL
identified in this study. The linked markers combined with GC enable rapid introgression of the
traits.
The non-GMO QTL identified in this work will allow development of parent lines and hybrids
with significantly reduced total saturated fatty acid content. Fine mapping and candidate gene
analyses for both lst and lpm are ongoing and will contribute to greater understanding of fatty
acid biosynthesis in oilseeds.
Key Words: Helianthus annuus, molecular marker, palmitic acid, quantitative trait loci, saturated fatty
acid, stearic acid
Introduction
The discovery of cytoplasmic male sterility and genes for fertility restoration allowed the production of
hybrid sunflower and promoted world sunflower production. The cultivated sunflower (Helianthus
annuus L.) is a major worldwide source of vegetable oil. Seed oil from cultivated sunflower is comprised
primarily of the saturated fatty acids palmitic (16:0) and stearic (18:0) acids, and the unsaturated fatty
acids oleic (18:1), linoleic (18:2) and linolenic (18:3) acids (Dorrel and Vick 1997). Sunflower oil is
premium oil because of its relatively high level of unsaturated fatty acid contents. Vegetable oils high in
unsaturated fatty acids, such as oleic and linoleic acids, may have the ability to lower plasma cholesterol,
reduce the risk of cardiovascular diseases (Mensink et al. 1994; Willett 1994). In contrast, saturated fatty
acids, such as palmitic and stearic acids, have been reported in certain studies to contribute to an increase
in the plasma cholesterol level, a factor in coronary heart disease (Willett 1994). Saturated fatty acids also
have higher melting points in general than unsaturated fatty acids of the same carbon number, which
contributes to cold tolerance problems in foodstuffs and can contribute to a waxy or greasy feel in the
mouth during ingestion. Despite sunflower oil already having a high level of unsaturated fatty acids, it is
still desirable to reduce its saturated fatty acid level for producing healthier oil. In addition, food products
made from fats and oils having less than 3.5% saturated fatty acids will typically contain less than 0.5
gram saturated fat per serving and as a result can be labeled as containing “zero saturated fat” under
current labeling regulations in the United States (www.fda.gov). Sunflower producing “zero saturated fat”
oil is not currently available in the market.
Palmitic and stearic acids are major saturated fatty acids in sunflower seeds and both are quantitatively
inherited. Several quantitative trait loci (QTL) for palmitic and stearic acids have been reported (PérezVich et al. 2002; Ebrahimi et al. 2008). Genetic background and environment also play significant roles in
palmitic and stearic acid inheritance (Pérez-Vich et al. 1999, 2002; Roche et al. 2006; Ebrahimi et al.
2008). Here, we present the results on the identification and characterization of genes conferring reduced
palmitic and stearic acid content in sunflower and the application of those genes in the development of
sunflowers producing unusually low saturated fatty acid oil.
Materials and Methods
Plant materials: Naturally occurring variants for low stearic acid (lst) and low palmitic acid (lpm)
were identified based on accumulated gas chromatography (GC) data from diverse Dow AgroSciences
(DAS) proprietary germplasm. These variants were independently crossed to develop F 2 mapping
populations. Two F2 populations were generated, one from cross ONN687R x H757B/LS10670B-B-173-23-5 (simplified as ONN687R/H757B in this report) and the other from cross H757B/LS10670B-17-323-5-4 x H280R[1]/687R-1-8-1 (simplified as H757B/H280R in this report). H757B and H280R were the
low stearic acid and low palmitic acid donors, respectively. Population ONN687R/H757B had 186 F2
individuals and was used to map low stearic acid QTLs, and population H757B/H280R had 188
individuals and was used to map low palmitic acid QTLs.
Fatty acid analyses: Fatty acid profiles of oils extracted from individual F 2 half-seeds were
quantified by gas chromatography (GC) and by using a proprietarily developed half-seed Fatty Acid
Methyl Ester (FAME) analysis method. Seven fatty acids were measured, including palmitic acid (16:0),
stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), arachidic acid (20:0), behenic acid (22:0) and
lignoceric acid (24:0). In brief, half-seeds were individually crushed with steal beads in 1 mL of heptane.
After centrifugation at 4000 rpm for 10 min, a 200 µL aliquot of the supernatant was transferred into a 2.5
mL auto-sampler vial, diluted with 300 µL heptane, and followed by the addition of 40 µL of 2M KOH
in methanol. The vials were capped and vortexed for 30 seconds. Samples were analyzed for fatty acid
contents on an Agilent 6890 GC-FID (Agilent Technologies) equipped with a SGE BPx70 30 meter x
0.25 mm ID column and 0.25 µm film thickness (SGE Analytical Science). The initial oven temperature
was 140 oC and maintained for one minute, followed by a rate increase of 20 oC /minute up to 250 oC.
The oven was held at this temp for 0.5 minutes. The inlet was set to split ratio of 1:2 and a temperature of
280 oC. A constant column flow rate of 2.0 mL/min helium was maintained throughout the run. The
detector was set to 300 oC with a constant carrier gas make up of 45.0 mL/min, fuel hydrogen flow of 30
mL/min, and oxidizer flow of 400 mL/min. An injection volume of 2 µL was used for all samples.
DNA extraction and SSR marker analyses: The rest of the F2 half-seeds left from fatty acid
analysis were grown in greenhouse. DNA was extracted from sampled leaf tissues. SSR markers were
used to genotype the mapping populations. PCR reactions were performed in GeneAmp PCR System
9700 (Applied Biosystems) with dual-384 well block. PCR reaction volume was 8 µL containing 10 ng of
DNA and 0.2 Units of Hot Start Taq DNA polymerase. The initial denaturing step was performed at 95
ºC for 12 minutes, followed by 40 cycles of 94 ºC for 5 seconds, 55 ºC for 15 seconds, 72 ºC for 30
seconds. The final extension was performed at 72 ºC for 30 minutes followed by 4 ºC forever. Forward
primers of SSR markers were end-labeled with fluorescent dyes FAM, HEX, or NED. Multiplexed PCR
products were resolved in ABI 3730XL DNA Analyzer.
QTL Mapping: Join Map 3.0 (Van Ooijen and Voorrips 2001) was used to create a genetic linkage
maps. Kosambi mapping function was employed (Kosambi 1944). MapQTL 4.0 (Van Ooijen et al. 2002)
was used to map QTLs. Interval mapping method (Jansen 1993) was employed.
Results and Discussions
Mapping of QTLs for low stearic and low palmitic acids: Parental lines ONN687R, H757B, and
H280R were screened with more than 400 SSR markers for identifying highly informative polymorphic
ones to genotype F2 mapping populations. All 186 individuals from the ONN687R/H757B F2 mapping
population were successfully genotyped with 76 selected informative polymorphic SSR markers and
evaluated for the seven fatty acids listed in Table 1. The total saturated fats for this population averaged
5.79% with the lowest plant containing 3.77% total saturated fats which was approaching the threshold
for United States Food and Drug Administration classification of a “zero saturated fat” oil (No Sat oil).
LOD scores associated with a P < 0.05 and P < 0.01 as estimated from a 1,000 iteration permutation test
(Churchill and Doerge 1993) for both the additive and additive + dominant statistical models were listed
in Table 2. As expected, the LOD scores for the additive model were lower than that for the additive +
dominant model as fewer parameters were estimated. One major QTL for low stearic acid was detected in
the HA1875-HA1865 interval on linkage group 17, spanning 21.8 cM (Fig. 1). It explained 36.1% of the
total phenotypic variation of stearic acid content in the population. Similarly, all 188 individuals from the
H757B/H280R F2 mapping population were successfully genotyped with 49 informative polymorphic
SSR markers and measured for all seven fatty acids, which resulted in the identification of one major
QTL for low palmitic acid (Fig. 1; detailed statistic analysis data not shown). The low palmitic acid QTL
was located in the 2 cM HA0908-HA0907 interval on linkage group 5 and explained 71.9% of the total
phenotypic variation in palmitic acid content in the mapping population with a LOD score of 3.0.
Table 1: Statistics on the ONN687R/H757B F2 mapping population (N= 186).
Fatty
Acid
Palmitic Acid (C16:0)
Stearic Acid (C18:0)
Oleic Acid (C18:1)
Linoleic Acid (C18:2)
Arachidic Acid (C20:0)
Behenic Acid (C22:0)
Lignoceric Acid (C24:0)
Total Saturates (C16-C18)
Total Saturates (C16-C24)
Mean
(%)
3.12
1.89
91.02
2.95
0.11
0.51
0.16
5.01
5.79
Standard
Deviation
0.64
0.63
4.74
4.43
0.15
0.43
0.15
0.87
0.88
Minimum
(%)
2.20
0.49
48.43
0.77
0.00
0.00
0.00
3.17
3.77
Maximum
(%)
4.94
3.47
95.39
44.26
0.64
1.31
0.56
7.87
8.08
Table 2: Experiment-wise permutation significance LOD score threshold.
Fatty acid
Fitting Trait as Additive Model
Fitting Trait as Additive and Dominant Model
Palmitic Acid
Stearic Acid
Oleic Acid
Linoleic Acid
Arachidic Acid
Behenic Acid
Lignoceric Acid
Total Saturates
(C16-C18)
Total Saturates
(C16-C24)
LOD Score (P<0.05/P<0.01)
2.5/3.8
2.5/3.2
1.7/2.0
1.6/1.9
2.4/3.1
2.5/3.1
2.5/3.3
2.5/3.4
LOD Score (P<0.05/P<0.01)
3.2/4.1
3.2/3.8
2.6/3.1
2.6/3.0
3.2/3.9
3.5/12.0
3.2/4.4
3.2/3.9
2.5/3.3
3.3/4.0
Figure 1: Showing the low stearic acid QTL in the HA1875-HA1865 interval on linkage group 17 and
the low palmitic acid QTL in the HA0908-HA0907 interval on linkage group 5. QTL intervals were filled
with grey color.
Development of reduced saturates sunflower: Seed fatty acid contents are complex traits in
sunflower. Many genes/QTLs and environment factors are involved in the inheritance. For example, the
dominant Ol mutation resulted in a significant increase of oleic acid content in sunflower seeds but seed
oleic content was significantly affected by genetic backgrounds (Soldatov 1976; Urie 1985; Miller et al.
1987; Schuppert et al. 2006). To develop sunflower germplasm with reduced saturates, a series of
hybridization were conducted to introgress and integrate the low stearic and low palmitic aicd QTLs
identified above into various genetic backgrounds. Normal breeding techniques such as backcross
breeding, mass and recurrent selections, and pedigree breeding, etc, were employed during breeding
practice. To speed up breeding process and increase selection gains, marker assisted selection was
employed. Flanking markers HA1875 and HA1865 were used for selection of the low stearic acid QTL,
and HA0907 and HA0908 for selection of the low palmitic acid QTL. Additional markers relatively
evenly distributed in the sunflower genome were also used to select the genetic background. In addition,
fatty acid profile of oils measured by GC was also used during the selection process. Successfully, a
series of sunflower lines producing unusually low levels of saturates have been developed at Dow
AgroSciences (Table 3). As can be seen in Table 3, progeny from crosses between lpm and lst
demonstrated total saturate levels as low as 2.33% in a high oleic (> 80%, Fad2-1) genetic background
and < 3.5% in a NuSun (55-50% oleic) background. Stearic acid levels reached as low as 0.25%, and
palmitic acid levels as low as 1.47%. Table 4 listed sunflower lines that had the lowest level of saturates
observed at Dow AgroSciences, 0.23% for low stearic acid, 1.37% for low palmitic acid and 2.33% for
total saturates. Interestingly, all three lowest levels occurred in the high oleic genetic background, which
suggested important roles of the high oleic mutation for the achievement of extremely low levels of
saturated fatty acids in sunflower seeds. The results demonstrated that the total saturated fatty acid
content of sunflower oil could be greatly reduced by introgression of the two QTLs identified in this study
into various genetic backgrounds such as high linoleic acid, medium oleic acid or high oleic acid
backgrounds, and that the linked SSR markers together with GC technology enabled rapid and efficient
introgression of the lst and lpm traits. The results also demonstrated the existence of interactions between
the two QTLs and their genetic backgrounds on seed saturated fatty acid level. To further understand the
genetic mechanisms of the lst and lpm QTLs, fine mapping and gene cloning of the two QTLs are
currently ongoing. More tightly linked molecular markers to the QTLs or gene specific markers
developed from the underlining genes could improve the efficiency of marker assisted selection during
breeding practice, and the cloned genes could benefit other oil crops for producing healthier oils.
Table 3: DAS sunflower germplasm with unusually low saturate levels (%).
Germplasm
NuSun/No Saturate
NS1982.16/OND163R-1-05
NS1982.8
No Saturate/High Oleic
NS1982.8-03
NS1982.8H117R[4]//H757B/LS10
670B///NS
1982.6-2-023-1-12-076
Low Saturate/Linoleic
CND117R/NS1982.8-3-06
OI1601B[2]//H757B/LS10670B[1]
//NS1982.6=B-3-04
CN2343B/4/CN2343B[2]//H757B/
LS10670B///NS1982.11#1#1-3-11
Low Stearic
NS1982.8/OND163R-2-12-009
H117R[4]//H757B/LS10670B///NS
1982.6-2-023-1-12-038
OID263R/NS1982.8-4-12-002
Low Palmitic
H251B[2]/IAST4=1=100//NS1982.16-11-39-041
NS1982.14-08
NS1982.16
Very High Oleic
H117R[4]//H757B/LS10670B/NS1
982.6-2-023-1-12-076
NS1982.8/OND163R-2-12-059
ON3351B/NS1982.8-1-04
C16:0
C18:0
C18:1
C18:2
Total Saturates
2.29
2.09
0.65
0.55
67.37
79.40
28.19
15.99
3.48
3.10
1.60
1.63
0.37
0.41
95.13
94.81
1.48
1.26
2.33
2.48
1.79
0.29
95.30
0.84
2.57
5.29
3.76
0.73
0.80
18.19
34.97
74.43
58.62
6.41
5.29
3.13
2.07
36.03
56.65
6.23
2.75
1.90
0.25
0.27
92.95
95.03
1.99
1.00
3.43
2.65
3.08
0.27
93.54
1.48
3.87
1.47
2.59
92.59
0.65
5.42
1.51
1.52
2.24
1.05
92.84
94.37
1.35
0.85
4.90
3.39
1.79
0.29
95.30
0.84
2.57
1.87
2.04
0.44
0.50
95.22
95.20
0.97
0.70
2.76
3.08
Table 4: DAS sunflower germplasm with lowest level (%) of stearic acid, palmitic acid and total
saturates.
Germplasm
NS1982.8/OND163R-12-90
H117R[4]//H757B/LS10670B-B-17-323=B1=2=16///NS1982.6-2-23.1-1
NS1982.8-03
C16:0
1.37
1.94
C18:0
1.70
0.23
C18:1
91.93
94.58
C18:2
2.83
1.80
Total Saturates
4.32
2.60
1.60
0.37
95.13
1.48
2.33
References
Churchill, G.A., and Doerge, R.W. 1994. Empirical threshold values for quantitative trait mapping.
Genetics 138: 963-971.
Dorrel, D.G., and Vick, B.A. 1997. Properties and processing of oilseed sunflower. p. 709- 746. In: A.A.
Schneiter (ed.), Sunflower technology and production. Crop Sci. Soc. Amer, Madison, WI, USA.
Ebrahimi, A., Maury, P., Berger, M., Grieu, P., and Sarrafi, A. 2008. QTL mapping of seed-quality traits
in sunflower recombinant inbred lines under different water regimes. Genome 51: 599-615.
Jansen, R.C. 1993. Interval mapping of multiple quantitative trait loci. Genetics 135: 205-211.
Kosambi, D.D. 1944. The estimation of map distances from recombination values. Ann Eug. 12:172-175.
Mensink, R.P., Temple, E.H.M., Hornstra, G. 1994. Dietary saturated and trans fatty acids and lipoprotein
metabolism. Ann. Med. 26: 461-464.
Miller, J.F., Zimmerman, D.C., and Vick, B.A. 1987. Genetic control of high oleic acid content in
sunflower oil. Crop Sci. 27: 923-926.
Pérez-Vich, B., Fernández-Martínez, J. M., Grondona, M., Knapp, S. J., and Berry, S. T. 2002. StearoylACP and oleoyl-PC desaturase genes cosegregate with quantitative trait loci underlying high stearic and
high oleic acid mutant phenotypes in sunflower. Theor. Appl. Genet. 104: 338-349.
Pérez-Vich, B., Garcés, R., and Fernández-Martínez, J.M. 1999. Epistatic interaction among loci
controlling the palmitic and the stearic acid levels in the seed oil of sunflower. Theor. Appl. Genet. 100:
105-111.
Roche, J., Bouniols, A., Mouloungui, Z., Barranco, T., and Cerny, M. 2006. Management of
environmental crop conditions to produce useful sunflower oil components. Eur. J. Lipid Sci. Technol.
108: 287-297.
Schuppert, G.F., Tang, S., Slabaugh, M.B., and Knapp, S.J. 2006. The sunflower high-oleic mutant Ol
carries variable tandem repeats of FAD2-1, a seed-specific oleoyl-phosphatidyl choline desaturase,
Molecular Breeding 17: 241–256.
Soldatov, K.I. 1976. Chemical mutagenesis in sunflower breeding. p. 352-357. In Proc. 7th Intl.
Sunflower Conf., Krasnodar, USSR (Vlaardingen, the Netherlands: Intl. Sunflower Assoc.).
Urie, L.A. 1985. Inheritance of high oleic acid in sunflower. Crop Sci. 25: 986-989.
Van Ooijen, J.W., and Voorrips, R.E. 2001. JoinMap Version 3.0, Software for the calculation of genetic
linkage maps. Plant Research International, Wageningen, the Netherlands.
Van Ooijen, J.W., Boer, M.P., Jansen, R.C., Maliepaard, C. 2002. MapQTL 4.0, Software for the
calculation of QTL positions on genetic maps. Plant Research International, Wageningen, the
Netherlands.
Willett, W.C. 1994. Diet and health: what should we eat? Science 264: 532-537.