Mol Breeding (2019) 39:79

https://doi.org/10.1007/s11032-019-0972-9

Novel alleles of FAD2-1A induce high levels of oleic acid

in soybean oil
Rachel Combs & Kristin Bilyeu

Received: 13 September 2018 / Accepted: 2 April 2019

# This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2019

Abstract Soybean plays an important role in seed oil
production for foods and industrial products in the USA.
Chemical hydrogenation of commodity soybean oil in-
creased functionality but unavoidably created trans fat
which has been linked to many health issues in humans.
An alternative to using hydrogenation of the oil to
enhance oxidative stability is to genetically increase
the level of oleic acid in the seed oil. Our goal was to
develop a soybean germplasm as a source for more
functional soybean oil with a high oleic acid, increased
stearic acid, and low linolenic acid profile utilizing new
and existing variant alleles of key fatty acid desaturase
genes. Using forward genetics, novel alleles of FAD2-
1A were discovered and characterized from a mutant
soybean population already containing elevated seed
stearic acid content. One of the two new FAD2-1A
alleles, when combined with existing mutant alleles of
FAD2-1B created a high oleic acid seed oil phenotype in
soybean germplasm lines. The other new FAD2-1A al-
lele produced lower and more variable oleic acid levels
when combined with existing mutant alleles of FAD2-
1B. Seed stearic acid increased to ~ 10–11% in lines
containing combinations of FAD2-1A and FAD2-1B
mutant alleles plus the SACPD-C missense mutant
R. Combs
Division of Biological Sciences, University of Missouri, 105
Tucker Hall, Columbia, MO 65211-7400, USA
K. Bilyeu (*)
Plant Genetics Research Unit, USDA-ARS, University of
Missouri, 110 Waters Hall, Columbia, MO 65211, USA
e-mail: Kristin.Bilyeu@ars.usda.gov
alleles; however, the increase in stearic acid was associ-
ated with a decrease in the oleic acid content and did not
meet the target of at least 20% stearic acid in the seed oil.
In addition, existing FAD3 mutant alleles were incorpo-
rated into the novel high oleic acid and high oleic/
elevated stearic acid soybean lines with four or five
targeted alleles combined (FAD2-1A, FAD2-1B, FAD3A,
FAD3C, and SACPD-C) to create soybean germplasm
with more functional soybean oil. This study is benefi-
cial for improving the quality of soybean oil based on
nutritional value and oxidative stability.
Keywords Soybean . Oil . Fatty acids . Oleic acid .
Stearic acid . Linolenic acid
Introduction
Soybean [Glycine max (L.) Merr.] is a primary source
for seed oil production in the USA. Because of its
function and widespread use in foods and industrial
products, it is important to genetically improve the fatty
acid profile of soybean oil to provide an alternative to
chemical hydrogenation. This hydrogenation process
unavoidably creates trans fat as a by-product which
has been linked to health problems in humans
(Crupkin and Zambelli 2008). Trans fats have been
mandated on food nutrition facts labels for over a de-
cade, and partially hydrogenated oils recently were
banned from foods in the USA (FDA 2003; FDA 2015).
Commodity soybean oil typically has a fatty acid pro-
file of 13% palmitic acid, 4% stearic acid, 20% oleic acid,
 79 Page 2 of 11
55% linoleic acid, and 8% linolenic acid (Fehr 2007;
Pham et al. 2010). Soybean oil with a high oleic acid
content over 70% and linolenic acid content less than 3%
is desirable for its oxidative stability and health benefits.
The delta-twelve fatty acid desaturase-2 (FAD2) enzyme
catalyzes the conversion of precursors of oleic acid (18:1)
into linoleic acid (18:2) (Pham et al. 2010). Previous
studies have shown that the two microsomal FAD2-1
desaturases, FAD2-1A (Glyma.10g278000) and FAD2-
1B (Glyma.20g111000), play an important role in oil
synthesis and the control of oleic acid level in developing
seeds (Fehr 2007; Pham et al. 2010). When mutant alleles
of these genes are combined, they produce higher levels of
oleic acid in the seed oil due to the reduction of activity in
the fatty acid desaturase enzyme, which leads to increased
accumulation of oleic acid (Haun et al. 2014; Hoshino
et al. 2010; Pham et al. 2011; Pham et al. 2010; Pham et al.
2012; Sweeney et al. 2017). Downregulation of FAD2-1A
and FAD2-1B is also the basis for the commercialized
Plenish® and Vistive® Gold soybean varieties with the
high oleic acid oil trait, while the Calyxt high oleic soy-
beans were developed from genome editing mutation of
FAD2-1A and FAD2-1B.
In addition to a higher content of oleic acid in soy-
bean oil, lower concentrations of the polyunsaturated
fatty acids, linoleic acid (18:2) and linolenic acid (18:3),
are desired because they have extremely low oxidative
stability. Studies have revealed that mutations in the
soybean microsomal omega-3 fatty acid desaturase
(FAD3) encoding genes contribute to a reduction in
linolenic acid levels (Bilyeu et al. 2005). Omega-3 fatty
acid desaturase enzymes catalyze the conversion of
linoleic acid precursors into linolenic acid precursors
by the incorporation of a third double bond in the carbon
chain. There are three FAD3 genes in soybean, and
studies have shown that FAD3A (Glyma.14 g194300)
in combination with FAD3B (Glyma.02g227200) or
FAD3C (Glyma.18g062000) reduced linolenic acid
levels in soybean oil to approximately 3% (Bilyeu
et al. 2011; Bilyeu et al. 2005). Less than 3% of linolenic
acid in the oil is the current market target to increase oil
functionality.
With a market demand for healthier foods that still
provide desired qualities such as texture from saturated
fats, there have been efforts to increase the stearic acid
content of soybean seed oil (Boersma et al. 2012;
Carrero-Colón et al. 2014; Gillman et al. 2014; Jeong
et al. 2018; Lakhssassi et al. 2017; Rahman et al. 1997;
Ruddle et al. 2013; Zhang et al. 2008). Increasing stearic
Mol Breeding (2019) 39:79
acid in the oil will increase the functionality of oil
without causing health issues, since stearic acid is
thought to be heart neutral (Crupkin and Zambelli
2008). However, stearic acid typically only makes up
about 3–4% of soybean oil. A single-nucleotide poly-
morphism (SNP) in the stearoyl-acyl carrier protein
desaturase C gene (SACPD-C; Glyma.14g121400) was
identified in a line (194D) derived from an ethyl
methanesulfonate (EMS)-induced Williams 82 mutant
population used for TILLING (Cooper et al. 2008;
Gillman et al. 2014). The SNP in SACPD-C, which
results in a missense amino acid substitution of an
almost invariant residue (V211E), showed elevated
levels of stearic acid in the seed oil (Gillman et al. 2014).
Soybean germplasm containing new combinations of
mutant fatty acid desaturase genes may provide sources
to develop soybean varieties that can improve the func-
tionality of soybean oil in the modern marketplace. We
identified and characterized novel mutant alleles of
FAD2-1A alone and in combination with an existing
mutant FAD2-1B gene and with mutant FAD3A and
FAD3C genes that produced high oleic (~ 80%)/low
linolenic (~ 3%) soybean germplasm (HOLL). Another
objective of this work was to develop soybean germ-
plasm that would reach homozygosity for the five mu-
tant alleles targeted: FAD2-1A, FAD2-1B, FAD3A,
FAD3C, and SACPD-C to attempt to create a high
stearic acid (> 20%) phenotype. Though the novel com-
bination of mutant alleles produced unique fatty acid
profiles of the seed oil, the target oil phenotype was not
achieved.
Materials and methods
Mutant population in 194D (Williams 82) background
Mutant lines were developed from an EMS treatment of
20 pounds of elevated stearic acid soybean line 194D
which itself was selected from an EMS mutagenesis of
BWilliams 82^ based on elevated seed stearic acid con-
tent (Gillman et al. 2014). Seeds were exposed to
50 mM EMS for 9 h before rinsing and field planting.
M1 plants were advanced to M3 seeds by a single seed
descent at the Costa Rica Seeds winter nursery near
Upala, Costa Rica; M3 seeds were planted at the Uni-
versity of Missouri South Farm Research Center in
Columbia, Missouri in 2014, and M3:4 seeds from single
plant threshes of 2431 plants were harvested.
Mol Breeding (2019) 39:79
Seed sampling for fatty acid phenotyping
Seeds were produced in three field seasons at the South
Farm Research Center in Columbia, Missouri. A three
seed composite from each M3:4 mutant lines from 2014
was analyzed for fatty acids in the oil with gas chroma-
tography as previously described (Beuselinck et al.
2006). The population was assessed to have a contam-
ination rate of less than 1% after identifying lines with
stearic acid less than 6% of the seed oil. Two lines were
identified (1478 and 1975) with elevated oleic acid
compared to the population that also had elevated stearic
acid. Three single seeds were subsequently analyzed
independently from the two original mutant lines. In
2015, seeds from the two mutant lines and control lines
were produced in the field and analyzed for fatty acids
as three seed composite samples from each of three
individual mutant plants for line 1975 but one plant for
line 1478. In 2015, the control lines 194D (a selection of
one mutagenized line from the population) and Williams
82 (W82) were produced as part of a separate study as
three plots of ten seeds each in a randomized complete
block design with seeds bulked from each plot. Williams
82 is considered wild-type for the alleles in fatty acid
biosynthesis. Line 194D was selected from a mutagen-
esis of Williams 82 for its elevated stearic acid and was
found to contain a V211E missense allele of the
SACPD-C gene. In 2016, the mutant lines and controls
were part of a field study also as three plots of ten seeds
each in a randomized complete block design with seeds
bulked from each plot.
FAD2-1A and FAD2-1B amplification and sequencing
DNA was isolated from the two mutant lines, named
1478 and 1975. Primers and PCR amplification for the
FAD2-1A and FAD2-1B genes were performed as ex-
plained by Pham et al. (2010). The PCR product was
confirmed by agarose gel electrophoresis, purified, and
Sanger sequenced by the DNA core center at the Uni-
versity of Missouri. Sequence alignments were analyzed
using a multiple sequence alignment from CLUSTALW
http://www.genome.jp/tools-bin/clustalw. Sequences
were aligned with Williams 82 reference to look for
nucleotide variations. Protein translation was done
using a six-frame translation http://www.bioline.
com/media/calculator/01_13.html and protein
alignment with CLUSTALW. Chromat files were also
Page 3 of 11 79
downloaded from the raw sequence data to analyze the
read at single base pairs.
Population development for allele combinations
The 1478 and 1975 lines were each crossed with KB14-
30#882 (FAD2-1AA, FAD2-1bb, and FAD3aacc) in the
summer of 2016 at South Farm Research Center in
Columbia, Missouri. Lines 1478 and 1975 were the
result of mutagenesis of the Williams 82 mutant line
194D identified to have elevated stearic acid due to a
missense mutation (V211E) in the SACPD-C gene in
addition to the novel FAD2-1A mutant alleles (Gillman
et al. 2014). In experimental line KB14-30#882, the
FAD2-1A alleles were wild-type and the FAD2-1B mu-
tant alleles contained the missense mutation P137R
from PI 283327 (Pham et al. 2010). For line KB14-
30#882, the FAD3A and FAD3C mutant alleles (splice
site and missense G128E, respectively) were derived
from the mutant line CX1512-44 (Bilyeu et al. 2005).
The F1 seeds from the resulting crosses, named KB16-
14 or KB16-15 for the parents 1478 or 1975 respective-
ly, were harvested and sent to the Costa Rica Seeds
winter nursery in Upala, Costa Rica to grow F1 plants,
followed by self-fertilization to produce F2 seeds after
one generation. Individual F2 seeds (225 from the 1478-
derived population and 325 from the 1975-derived pop-
ulation) were chipped to analyze the fatty acid profiles
by extracting seed oil from the chip for lipid gas chro-
matography (Beuselinck et al. 2006). The identified 13
or 18 F2 seeds produced in the Costa Rica environment
with a high oleic acid content (> 70%) from the KB16-
14 and KB16-15 populations, respectively, were germi-
nated in moist seed packets, and surviving seedlings
were transplanted and grown at South Farm Research
Center in Columbia, Missouri in the summer of 2017.
Not all seeds germinated or survived transplanting.
There were 12 F2 selected high oleic plants from
KB16-14 and 17 plants from KB16-15 that produced
F2:3 seeds. Following the harvest of single plants, five
single F2:3 seeds from each line were analyzed for fatty
acids by gas chromatography. Genotypes of the F2
plants were determined from DNA extracted from a
mixture of three F2:3 seeds (see below). For samples
that were segregating for one or more of the
target alleles, individual F3 seeds were also chipped for
gas chromatography, and the remainder of the seed was
utilized for DNA extraction and genotyping. Fatty acid
analysis was on single F3 seeds or chipped F3 seeds in
 79 Page 4 of 11
which DNA was made from the remaining seed section
to identify samples which had segregated to the desired
homozygous alleles by genotype assay. Single seed
samples from control plants that had all wild-type alleles
or just the SACPD-C V211E alleles were taken from
single plant threshes of Williams 82 or four independent
194D plants produced at South Farm Research Center in
Columbia, Missouri in the summer of 2017.
Genotyping and selection of soybean lines homozygous
for targeted mutant alleles
The selected new high oleic acid soybean lines were
genotyped for their fatty acid desaturase alleles after
harvest of F2:3 seeds, so no agronomic evaluations were
performed. One high oleic acid F2:3 line designated 53B
from the KB16-14 population had the following geno-
type: mutant alleles of FAD2-1A (C366R), FAD2-1B,
FAD3A, FAD3C, and SACPD-C. Five additional seeds
from this line were chipped for gas chromatography
following gas chromatography analysis of five single
F2:3 seeds from each line. Two high oleic acid F2 plants
from the KB16-14 population were identified by geno-
type to potentially produce F3 seeds for the high oleic/low
linolenic acid (HOLL) trait containing four homozygous
mutant genes. The genotype of line 100A was mutant
alleles of FAD2-1A (C366R), FAD2-1B, and FAD3A, and
segregating for FAD3C, whereas line 101A contained
mutant alleles of FAD2-1A (C366R), FAD2-1B, and
FAD3C, but was segregating for FAD3A. Twenty F3 seeds
of each line 100A and 101A were chipped for fatty acid
analysis, and a set of seven was selected with the lowest
linolenic acid levels for germination of the remainder of
the seed, transplanting to the field and generation of tissue
for DNA extraction. Of the surviving plants, DNA was
extracted, genotyping was performed, and four plants
(three from 100A and one from 101A) were confirmed
to be homozygous for the four targeted mutant alleles.
The fatty acid phenotypes of seed chips for the four F3
seeds with the mutant alleles of FAD2-1A (C366R),
FAD2-1B, FAD3A, and FAD3C were combined.
DNA extraction and genotype analysis
Genomic DNAwas isolated from a three seed mixture of
each selected high oleic acid F2:3 line using the DNeasy
Plant Mini Kit (Qiagen). For genotyping by sequence
analysis, DNAs were PCR amplified at 5–50 ng per
reaction for FAD2-1A as above (Pham et al. 2010) and
Mol Breeding (2019) 39:79
SACPD-C (Zhang et al. 2008). For SACPD-C, the
primers were 194Dsacpdexf: ACCCTGGGACAGAC
AACAAC and ZSACPDCrev: AGTCACAA
AGCCAAAAACCTG, and the 194Dsacpdexf primer
was used for sequencing. PCR products were verified by
agarose gel electrophoresis, followed by post-PCR pu-
rification. Samples were then Sanger sequenced at the
DNA Core Facility at the University of Missouri. Se-
quences were analyzed as described previously by
alignment with Williams 82 to assign genotypes for
FAD2-1A [wild-type (WT), C366R from 1478, or
T316I from 1975, and SACPD-C (WT or V211E)] at
the respective allele locations. The SNP in SACPD-C
from 194D was the thymine to adenine change 632 bp
from the start codon responsible for a V211E missense
mutation reported earlier (Gillman et al. 2014). Se-
quence traces were downloaded in Chromat files to
analyze clean reads and base pairs. A genotype of mu-
tant or wild-type was assigned according to the base pair
present at the allele location, but was assigned hetero-
zygous if both bases were present at that location in the
Chromat file. For some high oleic acid F2 plants, it was
determined that there were S117N mutant alleles segre-
gating (presumably from a parental donor plant of
KB14-30#882 that was wild-type and mutant S117N
heterozygous for FAD2-1A, even though the line was
thought to contain only wild-type FAD2-1A alleles)
(Dierking and Bilyeu 2009). Subsequent to this discov-
ery, the DNA of all F2:3 samples was analyzed by
SimpleProbe assay for the 17D S117N FAD2-1A alleles
(Pham et al. 2012). Soybean lines containing FAD2-1A
S117N alleles were eliminated from our analysis (one
from KB16-14 and six from KB16-15).
SimpleProbe assays were used for FAD3A and
FAD3C as described by Bilyeu et al. (Bilyeu et al.
2011), as well as FAD2-1B (P137) and FAD2-1A
(17D) as described by Pham et al. (Pham et al. 2012).
These genotyping assays were run using a Lightcycler
480 II real-time PCR instrument (Roche Applied Sci-
ences). Genotypes were assigned according to their
characteristic melting curves. The details of the assays
that distinguish the alleles used for sequence analysis
and genotyping are provided (Table 1).
Weblogo
Weblogo output of the amino acid conservation FAD2
enzyme was generated as part of the BLink feature at
NCBI (now retired) using GI number 197111722. The
Mol Breeding (2019) 39:79 Page 5 of 11 79

Table 1 Details of genotyping assays distinguishing variant alleles of fatty acid desaturase genes from their wild-type alleles

Gene Allele Primer F Primer R SimpleProbec

FAD2-1Aa C366R or T316I ACTGCATCGAATAA TGATATTGTCCCGT

and screening TACAAGCC GCAGC
FAD2-1Bb Screening CCCGCTGTCCCTTT TTACATTATAGCCA
TAAACT TGGATCGCTAC
SACPD-C V211E ACCCTGGGACAGAC AGTCACAAAGCCAA
AACAAC AAACCTG
FAD2-1A S117N CCAAGGTTGCCTTC TAGGCCACCCTATT GTACTTGCTGAAGG
TCACTGGT GTGAGTGTGAC CATGGTGA
FAD2-1B P137R GGTTCTCCAAGGTT AGGGTTGTTCAGGT AGTCCCTTATTTCT
GCATTCTTACT ACTTGGTGT CATGGAAAATAAGC
FAD3A Splice TTGCATCACCATGG AGCTATTATCTAGC GTTACCTTGCCGCG
TCATCAT ATTAACCTCA ATACCA
FAD3C G128E GTCCTTTGTTGAAC CTCCTGCAAAAAAT AGGAACCGACCATC
AGCATT CCATGAGTTGT CATGGTATGGTA
CAAGAAT
a
The new FAD2-1A alleles were discovered by sequencing PCR products. Distinguishing the C366R or T316I alleles from wild-type FAD2-
1A alleles was by sequencing PCR products
b
The FAD2-1B alleles were determined to be wild-type by sequencing PCR products
c
The genotyping assays based on SimpleProbe oligonucleotides, as indicated. In each of these cases, the 5′ (left) end of the oligonucleotide
contained a fluorescein dye and SimpleProbe chemistry and the 3′ (right) end of the oligonucleotide contained a phosphate to eliminate
enzymatic nucleotide extension during PCR

top 100 best-matched sequences were aligned and used
as input for sequence LOGO http://weblogo.berkeley.
edu/logo.cgi.
Statistics
Minitab statistics software was used for an ANOVA and
Fisher pairwise comparisons for genotypes in Table 3
with p < 0.05. The Student’s t test with p values less than
0.01 was used to determine significant differences in
mean fatty acid values for different genotypes for the
four and five allele combinations in Table 4.
Results
Discovery of novel missense alleles in FAD2-1A gene
in mutant soybean lines with increased oleic acid
Two mutant soybean lines with increased oleic acid in
the seed oil were identified in a forward genetics screen
of an elevated stearic acid mutant population fixed for a
missense V211E mutation in SACPD-C (Gillman et al.
2014). Seeds from lines 1478 and 1975 produced in the
field in 2014 exhibited a mean of 35.3% and 33.8%
oleic acid, respectively, in the seed oil compared to
19.3% oleic acid for the mutant population mean and
26.6% oleic acid for the control line Williams 82
(Table 2). There was no obvious segregation for oleic
acid content in lines 1478 or 1975. Seeds of lines 1478
and 1975 produced in the field in 2015 and 2016 also
contained increased levels of oleic acid in the seed oil
compared to a mutant control line with the SACPD-C
mutation (194D) and Williams 82.
The candidate genes for elevated oleic acid in soy-
bean seed oil were FAD2-1A and FAD2-1B. These genes
were amplified and sequenced from lines 1478 and
1975. Two novel missense alleles were found in the
FAD2-1A gene in lines 1478 and 1975. The SNPs were
identified from comparing sequence alignments to the
Williams 82 reference sequence. No new mutations
were found in FAD2-1B. The missense allele in the
1478 line was a cysteine to arginine amino acid change
at position 366 FAD2-1A (C366R) near the carboxyl
terminus of the protein from a thymine to cytosine
mutation at position 1096 of the coding sequence
(Fig. 1). The missense allele in the 1975 line was a
threonine to isoleucine amino acid change at position
316 FAD2-1A (T316I) from a cytosine to thymine mu-
tation at position 947 of the coding sequence (Fig. 1).
 79 Page 6 of 11 Mol Breeding (2019) 39:79

Table 2 Seed oil fatty acid composition of field-produced soybean mutants 1478, 1975, and control lines over 3 years

Year Line Sample Fatty acid composition (percent of oil)

Palmitic Stearic Oleic Linoleic Linolenic

2014 1478 3C (1)a 8.9 11.7 34.8 37.4 7.2

1478-1 1 9.0 13.2 36.5 34.4 6.9
1478-2 1 9.5 11.9 32.2 39.3 7.0
1478-3 1 8.5 14.3 37.5 33.1 6.6
1975 3C (1) 8.3 10.0 34.7 40.1 6.8
1975-1 1 8.4 11.2 31.4 42.2 6.8
1975-2 1 8.1 13.2 36.7 35.8 6.2
1975-3 1 9.7 9.8 32.3 41.6 6.6
194D pop. 3C (2431) 9.9 ± 0.6b 11.5 ± 1.8 19.3 ± 1.9 52.2 ± 2.5 7.2 ± 0.5
W82 3C (3) 11.1 ± 0.2 3.9 ± 0.2 26.6 ± 2.2 51.9 ± 1.8 6.4 ± 0.3
2015 1478 3C (1) 9.9 10.5 33.1 38.6 7.9
1975 3C (3) 8.9 ± 0.3 11.8 ± 0.3 35.2 ± 2.2 36.9 ± 1.8 7.2 ± 0.2
194D 3C (3) 10.1 ± 0.2 11.6 ± 1.0 19.7 ± 1.0 50.8 ± 0.2 7.9 ± 0.5
W82 3C (3) 11.6 ± 0.1 3.9 ± 0.1 24.5 ± 1.0 52.8 ± 0.6 7.2 ± 0.4
2016 1478 3C (3) 9.7 ± 0.2 10.9 ± 0.6 33.1 ± 1.6 39.1 ± 1.8 7.2 ± 0.4
1975 3C (3) 9.3 ± 0.1 11.2 ± 0.31 37.1 ± 4.1 36.0 ± 3.9 6.4 ± 0.61
194D 3C (3) 10.5 ± 0.1 10.0 ± 0.7 18.8 ± 1.3 53.2 ± 1.5 7.5 ± 0.2
W82 3C (3) 11 ± 0.1 3.9 ± 0.1 24.2 ± 2.9 53.9 ± 1.9 7.0 ± 0.9
a
The sampling method is noted to indicate fatty acid analysis for the number of whole seeds composited where 3C indicates three seeds
analyzed together followed by the number of replications in parenthesis, and 1 indicates a single seed was analyzed
b
The mean values are indicated followed by ±, the standard deviation of the mean for replicated samples

To examine the possible effect these alleles might
have on enzyme function, the amino acid conserva-
tion at the respective missense locations was ana-
lyzed. The neutral cysteine amino acid at position
366 of FAD2-1A is highly conserved, although it is
near a region of low conservation (amino acid posi-
tions 370–373). The arginine replacement in line
1478 is positively charged and polar (Fig. 1). The
neutral threonine amino acid at position 316 of
FAD2-1A changed to the neutral amino acid isoleu-
cine in line 1975 was found in the middle of the
highly conserved region II described to be critical
for enzyme function (Shanklin et al. 1994).
Assessment of combinations of novel alleles
with increased oleic acid
To evaluate the ability of the novel FAD2-1A alleles to
contribute to high oleic acid and high stearic acid oil
phenotypes, novel soybean germplasm was created for
two populations with the FAD2-1A allele and SACPD-C
mutant donor either line 1478 or 1975, and the other
parent an experimental line that contained mutant alleles
for elevated oleic acid and low linolenic acid in the seed
oil (Fig. 2). The process for population development
was to first produce F1 seeds that were heterozygous
for all five mutant alleles, where the FAD2-1A mutant
donor was either the C366R from line 1478 or the T316I
from line 1975. Lines 1478 and 1975 also contributed to
V211E mutant alleles of SACPD-C (Fig. 1). Seed chips
from individual F2 seeds produced in the winter nursery
were analyzed for the oleic acid levels, and seeds with
greater than 70% oleic acid were selected for growth and
genotype and phenotype characterization in our US field
environment. The fatty acid profile was subsequently
determined for fatty acids in F2:3 seeds. The experimen-
tal line KB14-30#882 parent contained wild-type alleles
of FAD2-1A and SACPD-C and homozygous mutant
alleles for FAD2-1B (P137R), FAD3A (splice), and
FAD3C (G128E) (Pham et al. 2010; Pham et al. 2012).
The other parent was either line 1478 with homozygous
mutant alleles for FAD2-1A (C66R) and SACPD-C
Mol Breeding (2019) 39:79 Page 7 of 11 79

W82. 310 KVFHHITDTHVAHHLFSTMPHYHAMEATNAIKPILGEYYQFDDTPFYKALWREARECLYV

a 1478.
1975.
310 KVFHHITDTHVAHHLFSTMPHYHAMEATNAIKPILGEYYQFDDTPFYKALWREARERLYV
310 KVFHHIIDTHVAHHLFSTMPHYHAMEATNAIKPILGEYYQFDDTPFYKALWREARECLYV
****** ************************************************* ***

c 1478

weblogo.berkeley.edu
1975

Fig. 1 Characterization of novel FAD2-1A missense alleles in in
soybean mutant lines 1478 and 1975. a Partial FAD2-1A amino
acid sequence alignment with reference Williams 82. Amino acid
position is indicated in front of the alignment. Identical amino
acids are highlighted in black and the mutations in lines 1975 and
1478 are indicated with no highlight. b Chromas DNA sequence
traces for the two mutations. The peaks correspond to the DNA
sequence information from the Sanger sequence analysis. The
positions of the DNA sequence change are indicated above the
trace by the red highlight. c Weblogo output of amino acid
weblogo.berkeley.edu
conservation for the FAD2-1A enzyme as part of the BLINK
feature at NCBI using GI no. 197111722. The top 100 best-
matched sequences were aligned and used as input for sequence
LOGO http://weblogo.berkeley.edu/logo.cgi. Each stack of
symbols represents a position in the amino acid sequence. The
height of the stack relates to the sequence conservation at the
corresponding location, and the individual symbol height
represents the relative frequency of the amino acids at that
location. The arrows point to the amino acid missense positions
as indicated in a box for line 1478 and 1975

(V211E) or line 1975 with homozygous mutant alleles
for FAD2-1A (T316I) and SACPD-C (V211E).
Within the two mutant population selections, a phe-
notypic comparison of field-produced F3 seeds was
conducted for the two novel mutant alleles of FAD2-
1A in combination with mutant alleles of FAD2-1B in
addition to the wild-type and mutant versions of
SACPD-C (Table 3). The status of the FAD3A and
FAD3C alleles was ignored for this analysis. Plants that
were confirmed by genotype to be homozygous mutant
for the FAD2-1A (either C366R or T316I) and FAD2-1B
(P137R) alleles and homozygous mutant (V211E) or
 79 Page 8 of 11
Fig. 2 Process for creating novel soybean germplasm segregating
for mutant alleles of five different genes involved in fatty acid
desaturation of the seed oil. New mutant lines 1478 and 1975 with
FAD2-1A and SACPD-C mutations were individually crossed with
the experimental line KB14-30#882 with mutant alleles of FAD2-
1B, FAD3A, and FAD3C. The two F2 populations were screened
for fatty acid phenotype to identify high oleic acid individual F2
seeds that were germinated and confirmed by genotype to be
homozygous for the novel combinations of mutations in FAD2-
1A and FAD2-1B. These high oleic acid F2 plants were segregating
for the other three genes, and a subset of those plants was identified
that contained different homozygous alleles of SACPD-C, FAD3A,
and FAD3C. Those plants were evaluated for their F2:3 seed fatty
acid phenotypes. Most of the lines were still segregating for
SACPD-C, FAD3A, or FAD3C, so additional genotyping was done
to identify individual seeds with homozygous genotypes
functional (WT) for the SACPD-C alleles were ana-
lyzed. Since there was no initial selection for the stearic
acid phenotype, many of the lines were still segregating
for the SACPD-C alleles, so some samples were chipped
F3 seeds in which the remainder of the seed was geno-
typed to determine the SACPD-C allele status. Lines
with mutant FAD2-1A and FAD2-1B alleles but wild-
type SACPD-C alleles are designated herein as HO,
while lines with mutant FAD2-1A, FAD2-1B, and
SACPD-C alleles are designated herein as HOS. Seeds
from lines containing the 1478 FAD2-1A C366R alleles
Mol Breeding (2019) 39:79
(1478-HO and 1478-HOS) had significantly higher
mean oleic acid seed oil content compared to lines
containing the 1975 T316I FAD2-1A alleles (1975-HO
and 1975-HOS) (Table 3). The 1478-derived lines that
contained the C366R alleles had an average of 22%
higher mean oleic acid in the seed oil than lines that
contained the T316I alleles. The 1975-HOS lines that
had the SACPD-C mutation had a significantly higher
mean stearic acid seed oil content compared to lines
from the 1478-HOS lines (~ 1.5%). For HOS lines com-
pared to HO lines with the same FAD2-1A alleles, there
was a significant increase in stearic acid and a signifi-
cant decrease in oleic acid that was approximately
equivalent. The mean sum of stearic acid and oleic acid
was not significantly different between HO and HOS
lines. The stearic acid level for the 1478-HOS lines was
not significantly different from the stearic acid level
from the control line 194D with the same SACPD-C
alleles, but the 1975-HOS stearic acid level was slightly
and significantly higher.
Development of new soybean germplasm with more
functional fatty acid profiles
One of the objectives of this work was to develop
soybean germplasm with the high oleic acid/low
linolenic acid (HOLL) trait and also the HOLL trait
with the high stearic acid trait. For the HOLL trait,
four mutant alleles were targeted, FAD2-1A, FAD2-
1B, FAD3A, and FAD3C; for the HOLL plus high
stearic acid trait, the four FAD alleles plus the
SACPD-C alleles were targeted. From F2:3 seeds, a
single plant (53B) from the 1478 FAD2-1A (C366R)
population was identified with the homozygous com-
bination of all five targeted alleles desired for the
HOLL plus high stearic acid trait. None of the F2
generation plants from the 1478 FAD2-1A (C366R)
population were homozygous for the four targeted
HOLL mutant genes and wild-type for SACPD-C.
However, phenotypic screening and genotypic selec-
tion of homozygous mutant FAD3A or FAD3C alleles
from two segregating F 2:3 plants from the 1478
FAD2-1A (C366R) population resulted in the identi-
fication of four seeds containing homozygous alleles
of the four targeted mutant FAD2 genes with wild-
type alleles of SACPD-C (designated 100/101A). The
fatty acid profile of the line 53B demonstrated a high
oleic acid and low linolenic acid phenotype (< 3%
linolenic acid), and stearic acid was 8.4% of the seed
Mol Breeding (2019) 39:79 Page 9 of 11 79

Table 3 Seed oil fatty acid composition of 2017 field-produced F3 seeds selected for different combinations of FAD2-1A, FAD2-1B, and
SACPD-C mutant alleles

Designation Alleles n= Fatty acid composition (percent of oil)

FAD2-1A FAD21-B SACPD-C Palmitic Stearic Oleic Linoleic Linolenic

1478-HO C366R P137R WT 20 (4)b 7.5 Dc 3.5 C 81.2 A 4.6 C 3.1 D

1478-HOS C366R P137R V211E 25 (7) 6.6 E 9.6 B 74.5 B 5.8 C 3.5 CD
1975-HO T316I P137R WT 26 (8) 8.3 C 3.9 C 60.3 C 23.3 B 4.2 C
1975-HOS T316I P137R V211E 32 (8) 8.0 C 11.1 A 52.3 D 22.3 B 6.3 B
194D WT WT V211E 20 9.7 B 10.1 B 20.1 F 52.0 A 8.0 A
W82 WT WT WT 5 11.0 A 4.8 C 24.9 E 52.6 A 6.6 B
a
The variant alleles are indicated by the missense position. For FAD2-1A, C366R was derived from mutant line 1478, T316I was derived
from mutant line 1975, and the P137R alleles of FAD2-1B were derived from the PI 283327. For SACPD-C, V211E alleles were derived
from the mutant parental line 194D. WT indicates functional alleles
b
The total number, n, of seeds analyzed is followed in parenthesis by the number of F2 plants used in the analysis. Some F2 plants produced
F3 seeds that had segregated into different genotype designations
c
The mean values are indicated followed by a letter of significance. Mean values within a column with different letters of significance were
significantly different (p = 0.05)

oil (Table 4). The fatty acid profile of the line 100/
101A selections also had a high oleic acid and low
linolenic acid phenotype, and stearic acid was 3.3%
(Table 4). The inclusion of homozygous selection for
mutant FAD3A and FAD3C alleles produced a mean
linolenic acid level of 2.3% of the seed oil when the
FAD2-1A C366R and FAD2-1B P137 alleles were
combined.
Discussion
Two novel missense mutation alleles of the soybean
FAD2-1A gene encoding an enzyme that converts
oleic acid precursors into linoleic acid precursors in
the seed oil were identified. The novel alleles were
discovered in a background of a mutant soybean line
containing elevated stearic acid in the seed oil due to
missense alleles of the SACPD-C gene. When com-
paring the two new FAD2-1A missense alleles, there
was an obvious distinction between the oleic acid
levels in the seed oil only after combining with mu-
tant FAD2-1B alleles. Lines containing the 1478
C366R FAD2-1A alleles had a higher mean oleic acid
seed oil content compared to lines containing the
1975 FAD2-1A allele by ~ 22% when combined with
mutant alleles of FAD2-1B. This result was not un-
precedented, since we and others have determined
that not all alleles of FAD2-1A or FAD2-1B have the
same effects on oleic acid levels, singly or in combi-
nation (Bilyeu et al. 2018; Hoshino et al. 2010; Pham
et al. 2011; Pham et al. 2010; Sweeney et al. 2017;
Thapa et al. 2016). The 1478 C366R FAD2-1A allele
caused an amino acid change that did not retain

Table 4 Seed oil fatty acid composition of 2017 field produced low linolenic acid soybean mutants 53B and 100/101A

Percent total oil

Line Genotype n= Palmitic Stearic Oleic Linoleic Linolenic

53B HOLLSa 10 6.8 ± 0.4 8.4 ± 1.2**b 75.8 ± 1.6** 6.7 ± 1.6* 2.3 ± 0.2
100/101A HOLL 4 6.6 ± 0.3 3.3 ± 0.3 83.4 ± 1.9 4.5 ± 1.3 2.2 ± 0.3
a
HOLLS indicates the homozygous allele combination FAD2-1A (C366R), FAD2-1B (P137R), FAD3A (splice), FAD3C (G128E), and
SACPD-C (V211E), while HOLL has the same genotype, except it was wild-type for SACPD-C
b
The mean values are indicated followed by ±, the standard deviation, and an * or ** is indicated if the mean values in the column differ at
p < 0.05 or 0.01significance, respectively
 79 Page 10 of 11
similar properties to the wild-type version; the 1975
T316I FAD2-1A amino acid substitution was in a
more conserved region, but the amino acid substitu-
tion was with one with similar properties. The allelic
series of FAD2-1A with FAD2-1B genes now avail-
able may provide additional information to under-
stand how structural features of these enzymes influ-
ence their biochemical activities.
Our results demonstrated that combining certain
mutant alleles of FAD2-1A with FAD2-1B genes pro-
duced a high oleic acid phenotype (> 70%) in the
1478 population containing the C366R FAD2-1A al-
leles plus the P137R FAD2-1B alleles. The oleic acid
phenotype in seed oil of this new soybean germplasm
was similar to the oleic acid phenotype of other
characterized high oleic acid soybean lines produced
in multiple environments (Bilyeu et al. 2018). As
expected, combining FAD3A and FAD3C homozy-
gous mutant alleles also reduced the linolenic acid,
to create HOLL lines, comparable with previous ex-
periments (Bilyeu et al. 2018; Pham et al. 2012). The
new soybean germplasm developed from line 100/
101A offers alternative opportunities to develop soy-
bean varieties with the high oleic acid/low linolenic
acid oil trait.
There have been reports of varying levels of stearic
acid for soybean lines combining the high oleic acid
alleles of FAD2-1A and FAD2-1B with mutant alleles
of SACPD-C. In one study, HOS combinations pro-
duced either 12.7% or 10.4% stearic acid in the high
oleic genetic background (Ruddle et al. 2014). In our
previous work, we characterized HOS lines that pro-
duced over 18% stearic acid in a high oleic acid
genetic background, but the stearic acid phenotype
was variable depending on the production environ-
ment (Bilyeu et al. 2018). One F2 plant (53B) from
our 1478 population was identified to be mutant for all
five alleles of interest: FAD2-1A, FAD2-1B, FAD3A,
FAD3C, and SACPD-C. Although this germplasm
contained less than the target stearic acid level, it still
produced a high oleic acid phenotype of ~ 76% oleic
acid with elevated stearic acid in the seed oil. The
functional properties of oil with elevated stearic acid
and high oleic acid plus low linolenic acid require
further characterization. It is possible that this unique
combination may provide desirable functionalities in
foods or for industrial uses. This germplasm will be
important for future studies on the advancement of
soybean oil functionality.
Mol Breeding (2019) 39:79
Soybean breeding for seed oil quality has a long
history with variation accessed through mutagenesis,
biotechnologies, and natural variation. The identifica-
tion of the causative alleles for seed oil traits shifts the
breeding scheme so that direct selection of those
alleles is potentially more efficient than phenotypic
selection during the breeding process. We routinely
develop segregating populations and use perfect mo-
lecular markers for genotypic selection of four to
eight alleles. Based on the expected inheritance ra-
tios, determining the appropriate population size and
fixing as many desired homozygous alleles as early
as possible is one strategy that has been successful.
For example, selection of all five alleles for high
oleic acid, low linolenic acid, and elevated stearic
acid from the quintuple heterozygote F1 would be 1/
1024 F2 samples, although fixing four of those genes
with a fifth one still segregating increases the ratio
slightly to 3/1024. Once the targeted alleles have
been fixed in different genetic backgrounds,
intercrossing those lines can produce populations that
segregate for other phenotypes without regard for the
oil quality trait.
In conclusion, this research demonstrates that using
traditional plant breeding to combine novel mutant al-
leles of FAD2-1A with FAD2-1B, FAD3A, and FAD3C
genes will result in elevated levels of oleic acid and
reduced levels of linolenic acid in the seed oil. In com-
bination with SACPD-C, stearic acid seed oil content is
elevated; however, the trade-off is that oleic acid levels
decrease. These findings are beneficial as they contrib-
ute to our knowledge and ability to enhance soybean oil
quality.
Acknowledgments Christine Cole and Paul Little provided ex-
cellent technical assistance for this research.
Funding information Funding for some of this project was
provided as a grant from the United Soybean Board.
Compliance with ethical standards
Disclaimer Mention of trade names or commercial products in
this publication is solely for the purpose of providing specific
information and does not imply recommendation or endorsement
by the US Department of Agriculture. USDA is an equal oppor-
tunity provider and employer.
Conflict of interest The authors declare that they have no con-
flict of interest.
Mol Breeding (2019) 39:79
References
Beuselinck PR, Sleper DA, Bilyeu KD (2006) An assessment of
phenotype selection for linolenic acid using genetic markers.
Crop Sci 46:747–750
Bilyeu K, Palavalli L, Sleper D, Beuselinck P (2005) Mutations in
soybean microsomal omega-3 fatty acid desaturase genes
reduce linolenic acid concentration in soybean seeds. Crop
Sci 45:1830–1836
Bilyeu K, Gillman JD, LeRoy AR (2011) Novel mutant allele
combinations produce soybeans containing 1% linolenic acid
in the seed oil. Crop Sci 51:259–264. https://doi.org/10.2135
/cropsci2010.01.0044
Bilyeu K, Skrabisova M, Allen D, Rajcan I, Palmquist DE, Gillen
A, Mian R, Jo H (2018) The interaction of the soybean seed
high oleic acid oil trait with other fatty acid modifications. J
Am Oil Chem Soc 95:39–49. https://doi.org/10.1002
/aocs.12025
Boersma JG, Gillman JD, Bilyeu KD, Ablett GR, Grainger C,
Rajcan I (2012) New mutations in a gene associated with
enhanced stearic acid levels in soybean seed. Crop Sci 52:
1736–1742. https://doi.org/10.2135/cropsci2011.08.0411
Carrero-Colón M, Abshire N, Sweeney D, Gaskin E, Hudson K
(2014) Mutations in SACPD-C result in a range of elevated
stearic acid concentration in soybean seed. PLoS One 9:
e97891. https://doi.org/10.1371/journal.pone.0097891
Cooper J, Till BJ, Laprot RG, Darlow MC, Kleffner JM, Jamai A,
El-Mellouki T, Liu S, Ritchie R, Nielsen N, Bilyeu KD,
Meksem K, Lomai L, Henikoff S (2008) TILLING to detect
induced mutations in soybean. BMC Plant Biol 8:9
Crupkin M, Zambelli A (2008) Detrimental impact of trans fats on
human health: stearic acid-rich fats as possible substitutes.
Compr Rev Food Sci Food Saf 7:271–279. https://doi.
org/10.1111/j.1541-4337.2008.00045.x
Dierking E, Bilyeu K (2009) New sources of soybean seed meal
and oil composition traits identified through TILLING. BMC
Plant Biol 9:89
FDA (2003) Food labeling: trans fatty acids in nutrition labeling,
nutrient content claims, and health claims vol 68 FR 41433.
Federal Register, Federal Register
FDA (2015) Final determination regarding partially hydrogenated
oils vol 80 FR 34651. Federal Register, Federal Register
Fehr WR (2007) Breeding for modified fatty acid composition in
soybean. Crop Sci 47:S–72-S-87. https://doi.org/10.2135
/cropsci2007.04.0004IPBS
Gillman JD, Stacey MG, Cui Y, Berg HR, Stacey G (2014)
Deletions of the SACPD-C locus elevate seed stearic acid
levels but also result in fatty acid and morphological alter-
ations in nitrogen fixing nodules. BMC Plant Biol 14:143.
https://doi.org/10.1186/1471-2229-14-143
Haun W, Coffman A, Clasen BM, Demorest ZL, Lowy A, Ray E,
Rettrath A, Stoddard T, Juillerat A, Cedrone F, Mathis L,
Voytas DF, Zhang F (2014) Improved soybean oil quality by
targeted mutagenesis of the fatty acid desaturase 2 gene
family. Plant Biotechnol J 12:934–940. https://doi.
org/10.1111/pbi.12201
Hoshino T, Takagi Y, Anai T (2010) Novel GmFAD2-1b mutant
alleles created by reverse genetics induce marked elevation of
oleic acid content in soybean seed in combination with
Page 11 of 11 79
GmFAD2-1a mutant alleles 60. https://doi.org/10.1270
/jsbbs.60.419
Jeong J-E, Krishnanand KP, Chang JH, Ha B-K, Kang ST, Bilyeu
K, Jo H, Song JT, Lee J-D (2018) A novel allele of
GmSACPD-C associated with high seed stearic acid concen-
tration in an EMS-induced mutant PE980 in soybean. Crop
Sci 58:192–203. https://doi.org/10.2135/cropsci2017.05.0313
Lakhssassi N, Colantonio V, Flowers ND, Zhou Z, Henry JS, Liu
S, Meksem K (2017) Stearoyl-acyl carrier protein desaturase
mutations uncover an impact of stearic acid in leaf and
nodule structure. Plant Physiol 174:1531–1543. https://doi.
org/10.1104/pp.16.01929
Pham A-T, Lee J-D, Shannon JG, Bilyeu K (2010) Mutant alleles
of FAD2-1A and FAD2-1B combine to produce soybeans
with the high oleic acid seed oil trait. BMC Plant Biol 10:195
Pham A-T, Lee J-D, Shannon J, Bilyeu K (2011) A novel FAD2-1
A allele in a soybean plant introduction offers an alternate
means to produce soybean seed oil with 85% oleic acid
content. TAG Theor Appl Genet 123:793–802. https://doi.
org/10.1007/s00122-011-1627-3
Pham AT, Shannon JG, Bilyeu KD (2012) Combinations of mu-
tant FAD2 and FAD3 genes to produce high oleic acid and
low linolenic acid soybean oil. Theor Appl Genet 125:503–
515
Rahman SM, Takagi Y, Kinoshita T (1997) Genetic control of high
stearic acid content in seed oil of two soybean mutants. Theor
Appl Genet 95:772–776. https://doi.org/10.1007
/s001220050624
Ruddle P, Whetten R, Cardinal A, Upchurch RG, Miranda L
(2013) Effect of a novel mutation in a Δ9-stearoyl-ACP-
desaturase on soybean seed oil composition. Theor Appl
Genet 126:241–249. https://doi.org/10.1007/s00122-012-
1977-5
Ruddle P, Whetten R, Cardinal A, Upchurch RG, Miranda L (2014)
Effect of Δ9-stearoyl-ACP-desaturase-C mutants in a high
oleic background on soybean seed oil composition. Theor
Appl Genet 127:349–358. https://doi.org/10.1007/s00122-
013-2223-5
Shanklin J, Whittle E, Fox BG (1994) Eight histidine residues are
catalytically essential in a membrane-associated iron enzyme,
stearoyl-CoA desaturase, and are conserved in alkane hy-
droxylase and xylene monooxygenase. Biochemistry 33:
12787–12794. https://doi.org/10.1021/bi00209a009
Sweeney DW, Carrero-Colón M, Hudson KA (2017)
Characterization of new allelic combinations for high-oleic
soybeans. Crop Sci 57:611–616. https://doi.org/10.2135
/cropsci2015.09.0596
Thapa R, Carrero-Colon M, Crowe M, Gaskin E, Hudson K
(2016) Novel FAD2–1A alleles confer an elevated oleic acid
phenotype in soybean seeds. Crop Sci 56:226–231.
https://doi.org/10.2135/cropsci2015.06.0339
Zhang P, Burton JW, Upchurch RG, Whittle E, Shanklin J, Dewey
RE (2008) Mutations in a Δ9–stearoyl-ACP-desaturase gene
are associated with enhanced stearic acid levels in soybean
seeds. Crop Sci 48:2305–2313. https://doi.org/10.2135
/cropsci2008.02.0084
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