Genome-Wide Association Analyses of Quantitative Disease Resistance in Diverse Sets of Soybean (Glycine Max (L.) Merr.) Plant Introductions

RESEARCH ARTICLE
Genome-wide association analyses of

quantitative disease resistance in diverse sets
of soybean [Glycine max (L.) Merr.] plant
introductions
William Rolling1, Rhiannon Lake2¤, Anne E. Dorrance1,3, Leah K. McHale ID1,2*
1 Center for Applied Plant Science and Center for Soybean Research, The Ohio State University, Columbus,
Ohio, United States of America, 2 Department of Horticulture and Crop Science, The Ohio State University,
a1111111111 Columbus, Ohio, United States of America, 3 Department of Plant Pathology, The Ohio State University,
a1111111111 Wooster, Ohio, United States of America
a1111111111
a1111111111 ¤ Current address: Corteva Agriscience, Napoleon, Ohio, United States of America
a1111111111 * [email protected]
Abstract
OPEN ACCESS Phytophthora sojae is one of the costliest soybean pathogens in the US. Quantitative dis-
Citation: Rolling W, Lake R, Dorrance AE, McHale ease resistance (QDR) is a vital part of Phytophthora disease management. In this study,
LK (2020) Genome-wide association analyses of QDR was measured in 478 and 495 plant introductions (PIs) towards P. sojae isolates
quantitative disease resistance in diverse sets of
soybean [Glycine max (L.) Merr.] plant
OH.121 and C2.S1, respectively, in genome-wide association (GWA) analyses to identify
introductions. PLoS ONE 15(3): e0227710. https:// genetic markers linked to QDR loci (QDRL). Populations were generated by sampling PIs
doi.org/10.1371/journal.pone.0227710 from the US, the Republic of Korea, and the full collection of PIs maintained by the USDA.
Editor: Istvan Rajcan, University of Guelph, Additionally, a meta-analysis of QDRL reported from bi-parental studies was done to com-
CANADA pare past and present findings. Twenty-four significant marker-trait associations were identi-
Received: August 29, 2019 fied from the 478 PIs phenotyped with OH.121, and an additional 24 marker-trait
Accepted: December 25, 2019
associations were identified from the 495 PIs phenotyped with C2.S1. In total, 48 significant
markers were distributed across 16 chromosomes and based on linkage analysis, represent
Published: March 20, 2020
a total of 44 QDRL. The majority of QDRL were identified with only one of the two isolates,
Copyright: © 2020 Rolling et al. This is an open and only a region on chromosome 13 was consistently identified. Regions on chromosomes
access article distributed under the terms of the
Creative Commons Attribution License, which
3, 13, and 17 were identified in previous GWA-analyses and were re-identified in this study.
permits unrestricted use, distribution, and Five QDRL co-localized with P. sojae meta-QDRL identified from QDRL reported in previous
reproduction in any medium, provided the original biparental mapping studies. The remaining regions represent novel QDRL, in the soybean-
author and source are credited.
P. sojae pathosystem and were primarily identified in germplasm from the Republic of
Data Availability Statement: All relevant data are Korea. Overall, the number of loci identified in this study highlights the complexity of QDR to
within the paper and its Supporting Information
P. sojae.
files.
Funding: This work was supported in part by an

award from the United Soybean Board to AD, an
award from the Ohio Soybean Council to LM, funds
from the Center for Applied Plant Sciences to WR,
AD & LM, state and federal funds appropriated to Introduction
The Ohio State University, College of Food,
Agricultural, and Environmental Sciences, and the Phytophthora root and stem rot [1] is an economically significant disease of soybean [Glycine
National Institute of Food and Agriculture, U.S. max (L.) Merr]. Infection occurs under favorable environmental conditions for disease
PLOS ONE | https://doi.org/10.1371/journal.pone.0227710 March 20, 2020 1 / 28

Genome-wide association analyses of quantitative disease resistance to Phytophthora sojae in soybean
Department of Agriculture Hatch projects for development, including saturated soils and temperatures around 25 ˚C [2–4]. Under these
Development of Disease Management Strategies conditions, zoospores of P. sojae chemotactically swim towards soybean roots [5,6]. Successful
for Soybean Pathogens in Ohio OHO01303 to AD,
infection of susceptible plants results in seed rot and damping off at early growth stages, and
and Genetic Analysis of Soybean Added-Value
Traits and Soybean Variety Development for Ohio wilting, stem lesions, and plant death at later growth stages. Genetic resistance is considered
OHO01279 to LM. the most effective strategy for preventing or reducing the impact of Phytophthora diseases
[4,7].
Competing interests: The authors have declared
that no competing interests exist. Soybean-breeding programs have relied on resistance conferred by single, dominantly
inherited Resistance to Phytophthora sojae (Rps)-genes. More than 30 Rps-genes/alleles have
been mapped (S1 Table) [8–25]. The effectiveness of Rps-mediated resistance is limited as
these genes only confer immunity towards specific races of P. sojae. Over time this results in
selection within pathogen populations, leading to adaptation [3,4] and limited Rps-gene life-
spans of 8–20 years [26]. A recent survey completed in the North Central region of the US
demonstrated that none of the deployed Rps-genes confer resistance to all P. sojae isolates,
with field populations having complex virulence patterns [27].
Quantitative disease resistance (QDR) is an additional form of genetic resistance, function-
ing through the action of multiple genes each conferring a portion of the overall phenotype
[28–30]. Though QDR does not confer complete immunity, the lack of race-specificity is pre-
dicted to place less selection on pathogen populations; thus, QDR is expected to be more dura-
ble than race-specific resistance [31–33]. For example, wheat growers have used QDR for
stripe and leaf rust resistance since the early 20th century, with no observed increase in patho-
gen virulence [34]. Similarly, QDR for the rice pathogen Magnaporthe oryzae contributed by
Pi21 has conferred effective resistance for over a century [35]. Used in combination, QDR can
also increase the durability of Resistance-gene (R-gene) mediated resistance as highlighted by
examples of pepper-potato virus Y [36] and root-knot nematode pathosystems of potato
[3,37].
In the soybean-P. sojae pathosystem, QDR has also played an important role in disease
resistance breeding [3]. QDR protects soybean yields when Rps-gene(s) are ineffective and the
environment is conducive to disease [7,38]. Breeding efforts to introduce QDR (and other
traits) for cultivar development involve large populations. Field testing can be misleading as
the pathotypes of P. sojae populations within fields are highly variable, the presence of an Rps-
gene could mask the effects of QDR [39], and QDR is not expressed within the first five days
following germination [40]. Marker-assisted selection (MAS) can provide an alternative
method for selecting cultivars with higher levels of QDR. Thus, studies to map the genetic posi-
tions of QDR can be applied to increase the efficiency of selection for these traits via MAS [41].
Numerous biparental mapping studies identified QDR loci (QDRL) towards P. sojae in US
cultivars Conrad, Sloan, and V71-370 [42–48]. Recently, the soybean nested associated map-
ping populations were used to study P. sojae resistance, identifying four QDRL [49]. In addi-
tion to US cultivars, QDRL have been mapped in landraces originating from China and the
Republic of Korea (South Korea, SK) using biparental mapping populations derived from
crosses with eight accessions [50–54]. The majority of these mapping studies identify several
QDRL within each population; most were small-effect loci explaining less than 10% of the vari-
ation (S2 Table).
Mapping studies have also been completed using genome-wide association (GWA) analy-
ses. This includes a study of 797 plant introductions (PIs) from SK [55] which had previously
been shown to be a source of Rps-genes and high levels of QDR against P. sojae [56], 175 Chi-
nese soybean breeding lines [57], and 279 breeding lines from the Yangtze-Huai Chinese
breeding program [58]. Recently, a collection of 169 Brazilian cultivars was used to identify
QDRL in regional germplasm [59]. Similar to loci identified by biparental mapping, these
GWA studies identified small-effect QDRL.

Although soybean growers deploy QDR to P. sojae in the US [3], US germplasm has never
been evaluated in a GWA analysis for QDR to P. sojae. A population derived from US germ-
plasm could be useful in identifying alleles in germplasm most adapted for the region. Though
alleles from adapted germplasm may be easier to introgress or maintain in elite cultivars, iden-
tifying novel QDRL from diverse germplasm remains a crucial aspect of resistance breeding.
Thus, the objective of the current research was to complete genome-wide association (GWA)
analyses by sampling US, SK, and the full diversity of the USDA soybean PI collection. Two
isolates were used to assay QDR because P. sojae is diverse [27] and previous studies have
mapped different QDRL depending on the isolate [47,48].
We phenotyped 547 (495 used in GWA analysis) PIs for QDR using isolate C2.S1 of P. sojae
and also measured QDR in a different set of 549 PIs (478 used in GWA analysis) with the sec-
ond isolate OH.12108.6.3 (OH.121) of P. sojae. In total, 48 SNPs associated with QDR to P.
sojae were identified and consolidated into 44 QDRL, with 21 QDRL identified with OH.121
and 23 QDRL identified with C2.S1. In addition to GWA analyses, we completed a meta-
QDRL analysis to consolidate previous results and identify any colocalization between the loci
identified in this study by GWA analysis and those QDR previously identified from biparental
mapping studies. The 44 QDRL reported here represent a mixture of both novel QDRL and
those that confirm results of previous mapping studies of this complex pathosystem.
Materials and methods

Genotypic data
We used publically available genotypic data from the SoySNP50K iSelect BeadChip [60] down-
loaded from Soybase [61] (www.soybase.org/snps). In total, 42,509 SNP data were available for
20,087 PIs. Monomorphic markers, markers with > 5% missing data, and markers with a
minor allele frequency of < 5% were removed prior to analyses. For the remaining SNPs, miss-
ing marker data was imputed with fastPHASE [62]. Using the 1096 PIs phenotyped in this
study, genome-wide linkage disequilibrium (LD) decay was estimated for both euchromatic
and heterochromatic regions by plotting physical distance vs. linkage (r2).
Germplasm diversity and selection

FastStructure [63] population analysis using default settings was used to identify related popu-
lations. PIs were assigned to populations using a membership coefficient threshold (Q-value)
of � 0.7. We sampled the USDA collection to create two distinct sets of PIs, phenotyped with
either P. sojae isolate OH.121 (OH set) or C2.S1 (C2 set). PIs were represented once and were
phenotyped with only C2.S1 or OH.121, not both isolates of P. sojae because a limited number
of seeds were provided by the germplasm center, limiting the assays to one isolate per PI.
Three populations were represented within the C2 set and OH set. The first populations were
randomly sampled twice from the 20,087 PIs to create the C2-GRIN and OH-GRIN popula-
tions. Population structure analyses identified a population of 817 PIs primarily consisting of
US-North-Central region PIs and 1805 PIs mainly originating from SK. The PIs originating
from the US were randomly sampled twice to create the C2-US and OH-US populations. Simi-
larly, the SK originating PIs were randomly sampled twice to create the C2-SK and OH-SK
populations.
For the C2 set of PIs (consisting of the C2-US, C2-SK, and C2-GRIN populations), 547 PIs
were phenotyped, and 495 PIs used for GWA analysis (S3 Table). The three populations had
samplings of 180 PIs for the US population, 162 PIs for the SK population, and 153 PIs sam-
pled from the GRIN population. The OH set of PIs (consisting of the OH-US, OH-SK, and
OH-GRIN populations) had 549 PIs, which were unique from those included in the C2 set.

The OH set included 478 PIs (S4 Table) in GWA analysis composed of 170 US PIs, 158 SK PIs,
and 150 PIs sampled from the GRIN population.
In addition to the PIs, cultivars with characterized QDR against P. sojae (‘L83-570—Rps3a’,
‘Williams 79—Rps1c’, ‘Conrad—rps’, ‘Sloan—rps’, ‘Resnik—Rps1k’,‘OX20-8—Rps1a’), were
included as checks. The P. sojae isolates were expected to be virulent on these lines despite the
presence of Rps-genes, with the exception of OH.121 on ‘Williams 79—Rps1c’. Before disease
assays, all seeds were surface sterilized using chlorine gas [64].
Quantitative disease resistance assays

Phenotyping of QDR was completed using the layer test [65] between February and April 2018
with 16 hrs of supplemental lighting and temperatures maintained between 18˚C and 26˚C.
Briefly, a 950mL Styrofoam cup was filled from bottom to top with: (1) 2.5 cm layer of coarse
vermiculite, (2) 7.6 cm of fine vermiculite, (3) a two-week-old culture of P. sojae grown on
dilute lima bean agar in a 100 mm x 15 mm Petri dish or for non-inoculated treatments 0.6 cm
of fine vermiculite, (4) 3.8 cm of fine vermiculite, (5) 8 seeds, and (6) a final layer of coarse ver-
miculite to cover the seeds. Each block of the experiment was maintained over a three-week
period during which the cups were watered twice a day.
The data collected for GWA analyses included an Inoculated Root Rot Score (IRRS), Inocu-
lated Root Weight (IRW), Inoculated Shoot Weight (ISW), Inoculated Plant Height (IPH),
Non-inoculated Root Weight, Shoot Weight and Plant Height (NRW, NSW, NPH). The plant
heights (IPH, NPH) were collected from three representative plants and averaged. The IRRS
score was scored on a scale of 1–9 [65]. Change in root and shoot weight (ΔRW, ΔSW) were
calculated by subtracting non-inoculated weights from inoculated weights for each PI line.
We used an incomplete block design with PIs randomized with restriction so that each
block contained an inoculated and non-inoculated experimental unit (Styrofoam cup) for each
PI represented in a block. Each block consisted of ~183 PIs, with 8 seeds per experimental
unit. Check cultivars were included in all blocks. Two replicates were completed for each PI
resulting in six blocks for both the C2 set and OH set, or 12 blocks total.
Best Linear Unbiased Predictor (BLUP) values and Best Linear Unbiased Estimators
(BLUEs) were calculated for PIs and checks, respectively, for each trait measured in the experi-
ment using the R package [66] lme4 [67]. The equation used for each trait was Yhijk = μ + Ch +
L(C)hi + B(R)jk + εhijk, where μ is the grand mean, Ch is the effect hth class representing one of
the six checks or an experimental PI, L(C)hi is the genotypic effect of each line, B(R)jk is the jth
block effect within kth rep, and ε represents the residual. Broad-sense heritability (H2) was cal-
culated for each trait H2 = σg2/(σg2 + σε2/r), where σg2 (genetic variance), σε2 (error variance),
and r (number of replicates).
Phytophthora isolates and Rps-gene assays

The focus of this study was on QDR, therefore isolates with complex virulence pathotypes
were used to limit Rps-gene responses masking QDR. The Ohio isolates OH.12108.6.3
(OH.121) (vir 1a, 1b, 1d, 1k, 2, 3a, 3c, 4, 5, 6, 7, 8) and C2.S1 (vir 1a, 1b, 1c, 1d, 1k, 2, 3a, 3c, 4,
5, 6, 7, 8) were used in the QDR assays. The virulence of these isolates had previously been
assessed [55], but to ensure the pathotypes were consistent after storage, virulence was reas-
sessed here.
To ensure that we were evaluating QDR and not Rps-mediated resistance, we evaluated the
547 lines in the C2 set and the 549 lines from the OH set with the hypocotyl test. Due to limited
seed number, eight seedlings were grown and two replicates completed. Scoring thresholds of
70% and 30% were applied. When no lesion developed on 70% of the plants, it was considered

a Rps-gene response and removed from GWA analyses. PIs that developed a lesion 70–90% of
the time were further scrutinized by checking the scores in the layer test to ensure a lack of an
Rps-gene response, and PIs with IRRS scores less than 1.5 were removed from the GWA
analyses.
GWA analyses
The C2 set and the OH set were each analyzed separately; four GWA analyses were completed
for C2 and OH set, for a total of 8. An analysis was completed using all PIs for a set as well as
the three populations (C2-US, C2-SK, C2-GRIN, OH-US, OH-SK, and OH-GRIN) that com-
prise each set. GWA analyses were implemented using the R [66] package GAPIT [68] using a
multiple locus mixed model (MLMM). General linear model, mixed linear model, compressed
mixed linear model, and FarmCPU were also tested, but did not yield appropriate Q-Q plots
or were not as able to resolve significant associations (S1 Fig). When implemented in GAPIT,
MLMM does not readily provide an estimate of percent variation explained. Instead, PVE was
estimated using CMLM. The Q-matrix resulting from fastStructure analyses was used as a
covariate. The significance threshold was determined using a modified Bonferroni adjustment
by calculating the Meff using SimpleM [69]. Genome-wide significance thresholds were deter-
mined by α/Meff where α = 0.05.
Haploview [70] was used for haplotype block assembly using the four-gamete rule [71]. A
haplotype block possessing a marker(s) with a significant marker-trait association was consid-
ered a single QDRL. Haplotype blocks for markers that were unlinked to other markers were
delimited according to the positions of the flanking markers.
Meta-QDRL analysis
We collected pertinent information for reported QDRL towards P. sojae identified prior to Jan
1, 2019. Collected data included (1) linkage maps, (2) R2 of genetic effect, (3) significance
(LOD), (4) linkage group, (5) confidence interval (CI), (6) predicted position of QDRL, (7)
generation and (8) size of populations from 16 biparental mapping studies (S2 Table). Due to
inconsistent reporting of these values, estimates were made for LOD values and confidence
intervals (CIs) when not provided. LOD values were calculated using the formula LOD = R2/
1.5. If the CI was not provided it was calculated using the equation CI = 163/(N x R2) where N
is the population size [72].
Meta-analysis was completed using the software BioMercator V3.1 [73,74]. The initial step
was to create a project-specific consensus map from genetic maps found in the biparental stud-
ies; this was aided by the inclusion of the consensus map [75] downloaded from Soybase [61].
QDRL were projected onto the project-specific consensus map. Studies prior to 2010 were
removed and only one mapping study per generation of each biparental cross was included.
The projected QDRL were used to identify position and CI based on Akaike-type criteria val-
ues. The physical positions of meta-QDRL were determined by selecting the markers flanking
the meta-QDRL and locating the positions of these markers in the Wms.82 genome (Wms.82.
a2.v1) [76].
Results
Population structure analysis and linkage disequilibrium
The population structure of the USDA germplasm collection was analyzed in order to generate
population-groups from which our GWA populations would be sampled. Population structure
analyses of the 20,087 PIs with SoySNP50K genotypic data resulted in marginal likelihood

estimations increasing with model complexities (number of population-groups) from 1 to 20

(S5 Table). A local maximum was present at k = 12, describing twelve major population-
groups of soybean PIs (Fig 1A).
Two population-groups were identified that primarily consisted of PIs originating from the
North Central US and SK, these were specifically sampled. PIs originating from SK have previ-
ously been shown to be a rich source of QDR alleles [55,56], providing novel QDR alleles to
breeders that can greatly benefit resistance breeding [30]. The US population-group is a poten-
tial source of QDR alleles in genetic backgrounds adapted to the North Central regions of the
US or alleles already deployed in US cultivars. To generate the six populations for our GWA
analyses, we sampled the SK population-group to create the C2-SK and OH-SK populations and
sampled the US population-group to create the C2-US and OH-US populations. Finally, the full
USDA germplasm collection was sampled to generate the C2-GRIN and OH-GRIN populations
to access a broader diversity, potentially providing novel, under-utilized QDR alleles.
The combination of the C2-SK, C2-US, and C2-GRIN populations were referred to as the
C2 set. Likewise, the OH set was comprised of the OH-SK, OH-US, and OH-GRIN popula-
tions. Eleven of the 12 population-groups identified by the fastStructure analysis of the 20,087
Fig 1. Estimated population structure of soybean plant introductions (PIs) genotyped with the SoySNP50K iSelect BeadChip. (A) Population
structure analysis of the 20,087 genotyped PIs maintained by the USDA Soybean Germplasm Center (Urbana, IL) was carried out at k = 12. (B) The C2
set consists of 495 PIs phenotyped with P. sojae isolate C2.S1. (C) The OH set consists of 478 PIs phenotyped with P. sojae isolate OH.121. The 12
population-groups identified by fastStructure are indicated by lower case letters. The United States (US) and South Korean (SK) population-groups of
interest are indicated with labels “b” and “j”, respectively. To create C2-US, C2-SK, OH-US, and OH-SK, the US and SK populations-groups (b, j) were
sampled.
https://doi.org/10.1371/journal.pone.0227710.g001

PIs in the germplasm collection were represented in both the C2 set and OH set (Fig 1B and
1C). Additional population structure analyses were completed to identify subpopulations in
the PIs comprising C2-US and OH-US as well as the C2-SK and OH-SK populations. These
analyses identified six and nine subpopulations within the US and SK populations, respectively
(S2 and S3 Figs). The majority of the subpopulations are present in the respective C2-US and
OH-US populations and C2-SK and OH-SK populations, with only slightly different propor-
tions of each subpopulation represented (S2 and S3 Figs).
A total of 33,234 (C2 set) and 34,248 (OH set) SNP-markers were used in GWA analyses.
There was an average density of one marker every 29 kb for both the C2 and OH sets. Due to
linkage disequilibrium (LD), SNP-markers could be consolidated to an effective marker num-
ber [69] of approximately 11,000 in the OH set and C2 set, as well as the OH-GRIN and
C2-GRIN populations. The effective marker numbers were nearly 5,000 for the C2-SK and
OH-SK populations, 4,051 in C2-US population, and 3,291 in the OH-US population (S4 Fig).
In the combined 974 individuals from the C2 and OH sets, where LD is expected to be lower
than the less diverse populations (i.e., C2-US, C2-SK, OH-US, and OH-SK) [77], LD decayed
to an r2 of 0.2, a common threshold of significant LD [78], at an approximate distance of
350kb in euchromatic regions and greater than 500kb in heterochromatic regions (S4 Fig).
Given that each population has an estimated marker density < 300 kb/marker, we anticipate
that the majority of the soybean genome was captured in each GWA analysis (S4 Fig).
Quantitative disease resistance of PIs in the C2 set

A total of 547 PIs were phenotyped with isolate C2.S1, of which 52 were subsequently removed
from analyses due to an Rps-gene response or missing data. For the remaining 495 PIs that
comprised the C2 set, best linear unbiased predictor (BLUP) values were calculated from the
raw measurements of inoculated and non-inoculated traits. The BLUP values for NRW, NSW,
and NPH had normal distributions, while IRRS, IRW, and ISW had nearly normal distribu-
tions with a slight positive skew and ΔRW, ΔSW and NPH had nearly normal distributions
with a slight negative skew (S5 Fig). For the C2 set, all of the inoculated traits had significant
genetic variance and moderate to moderately high broad-sense heritability, ranging from 0.52
for ΔRW to 0.78 in IRW (Table 1).
Within the C2 set, the C2-US, C2-SK, and C2-GRIN populations had comparable average
BLUP scores for each trait (S6 Fig). Despite similar means, subtle trends in the distributions of
BLUP values were present; the US population had significantly higher BLUP values for NRW
and NSW, indicating that these PIs were larger (S6 Fig). Consistent with previous studies, the
C2-SK population had the lowest BLUP values for IRRS (most resistant) and highest BLUP
values for IRW, ISW, and IPH of the three populations, once again demonstrating the high
resistance levels found in SK germplasm. The SK and GRIN populations had greater genetic
variance and a larger standard deviation around the means for all traits (S6 Fig). The C2-US
population had the lowest average heritability (H2 = 0.39) and genetic variance, though still
significant for all traits except ΔRW and ΔSW. Both the C2-SK and C2-GRIN populations also
had significant genetic variance for all traits and higher average broad-sense heritability
between 0.73 and 0.66, respectively (S6 Table).
In the C2 set, there was significant correlation between nearly all traits (Table 2). Correla-
tions were strongest among traits of the same type (inoculated, non-inoculated, or combined).
Correlations among inoculated traits were moderate to high, ranging from -0.53 for IRRS
(lower score is more resistant) and IPH to 0.87 for ISW and IRW. The correlation between the
combined traits ΔSW and ΔRW was also high at 0.79, and correlations among non-inoculated
traits were moderate to high, ranging from 0.59 for NPH and NRW to 0.83 for NSW and

Table 1. Heritability and genetic variance for layer test traits with Phytophthora sojae isolates C.2.S.1 and OH.121
for the C2 and OH sets of plant introductions, respectively.
C2 set OH set
a 2b 2c 2
Trait H σg H σg2
�� d
IRRS 0.58 0.94 0.65 1.98�
��
IRW 0.78 0.19 0.45 0.10�
��
ISW 0.65 0.23 0.68 0.27��
��
IPH 0.58 7.70 0.77 14.00��
ΔRW 0.52 0.11 ��
0.38 0.09��
ΔSW 0.56 0.12�� 0.43 0.16��
NRW 0.75 0.23�� 0.39 0.14�
��
NSW 0.66 0.25 0.65 0.30��
��
NPH 0.55 6.57 0.63 8.12��
a
, IRRS, inoculated root rot score; IRW, inoculated root weight; ISW, inoculated shoot weight; IPH, inoculated plant
height; ΔRW, change in root weight; ΔSW, change in shoot weight; NRW, non-inoculated root weight; NSW, non-
inoculated shoot weight; NPH, non-inoculated plant height
b
, H2, broad-sense heritability
c
, σg2, genetic variance;
d �
, , significant at P value < 0.05;
��
, significant at P value < 0.001.
https://doi.org/10.1371/journal.pone.0227710.t001
NRW. For the most part, correlations between different types of traits were low to moderate,
ranging from -0.21 for ΔRW and NPH to 0.56 for ISW and NSW. However, IRRS was not sig-
nificantly correlated to the non-inoculated traits, indicating that IRRS is independent of non-
inoculated plant size and weights.
Quantitative disease resistance of PIs in the OH set

A total of 549 PIs were phenotyped with isolate OH.121, and 71 PIs were removed from subse-
quent analyses due an Rps-gene response or missing data. Thus, the final OH set consisted of
Table 2. Significant correlations between layer test traits assessed within the C2 set (unshaded) and OH sets (shaded) of plant introductions.
Inoculated Combined Non-inoculated
Traita IRRS IRW ISW IPH ΔRW ΔSW NRW NSW NPH
b
IRRS 0.63 -0.54 -0.53 -0.57 -0.59 -- -- --
IRW -0.71 0.87 0.73 0.38 0.37 0.48 0.51 0.38
ISW -0.65 0.83 0.75 0.30 0.43 0.45 0.56 0.36
IPH -0.77 0.76 0.72 0.23 0.31 0.37 0.41 0.43
ΔRW -0.38 0.38 0.26 0.25 0.79 -0.43 -0.31 -0.21
ΔSW -0.51 0.44 0.48 0.39 0.58 -0.27 -0.35 -0.24
NRW -- 0.41 0.36 0.36 -0.45 -0.43 0.83 0.59
NSW -- 0.40 0.51 0.34 -0.30 -0.49 0.78 0.69
NPH -- 0.33 0.3 0.43 -0.19 -0.35 0.63 0.65
All displayed correlations were significant at a P value of < 0.001

a
, IRRS, inoculated root rot score; IRW, inoculated root weight; ISW, inoculated shoot weight; IPH, inoculated plant height; ΔRW, change in root weight; ΔSW, change
in shoot weight; NRW, non-inoculated root weight; NSW, non-inoculated shoot weight; NPH, non-inoculated plant height
b
, no significant correlation.

478 PIs. The BLUP values for NRW and NSW had normal distributions (S7 Fig). The other
traits had nearly normal distributions with a slight positive skew to IRRS, IRW, and ISW; a
slight negative skew for IPH and NPH; and heavy tailing in the ΔRW and ΔSW traits (S7 Fig).
All nine traits had significant genetic variance (Table 1) with moderate to high heritability,
ranging from 0.38 for ΔRW to 0.77 for IPH. Both the heritability and genetic variance were
comparable between isolates, with OH.121 traits on average having slightly higher genetic vari-
ance, higher residual variance, and marginally lower heritability (Table 1).
The average BLUP values of the three populations comprising the OH set (OH-GRIN,
OH-SK, OH-US) were very similar for each trait, with a greater standard deviation of BLUP
values in the OH-GRIN and OH-SK populations relative to the OH-US population (S7 Fig).
Genetic variance was lowest in the US population, and heritability was highest in the SK popu-
lation. The US and SK populations had similar BLUP values for IRRS, IRW, ISW, and IPH
and were significantly more resistant than the GRIN population. The US population had sig-
nificantly higher BLUP values for the non-inoculated traits (sans NSW compared to SK) indi-
cating, once again, that the US population averaged larger plants than the SK and GRIN
populations.
There were significant correlations among nearly all traits in the OH set (Table 2). Correla-
tions were strongest among traits of the same type (inoculated, non-inoculated, or combined).
Correlations among inoculated traits were moderate to high, ranging from -0.65 for IRRS and
ISW to 0.83 for ISW and IRW. The correlation between the combined traits ΔSW and ΔRW
was moderate at 0.58, and correlations among non-inoculated traits were moderate to high,
ranging from 0.63 for NPH and NRW to 0.78 for NSW and NRW. For the most part, correla-
tions between different types of traits were low to moderate, ranging from -0.19 for ΔRW and
NPH to -0.51 for ISW and NPH. However, similar to data from the C2 set, IRRS was not sig-
nificantly correlated to the non-inoculated traits, indicating that IRRS is independent of non-
inoculated plant size and weights.
Genome-wide association analyses

A total of 48 significant marker-traits associations were identified on 16 chromosomes (Tables
3 and 4; Figs 2 and 3). Markers in LD with significant markers were considered the region
most likely to contain the allele contributing to QDR and were used to delimit QDRL. Some
markers were associated with more than one trait or were within the same haplotype block;
therefore, the 48 marker-trait associations were consolidated into 44 QDRL.
GWA analyses of the C2 set, as well as C2-SK, C2-US, and C2-GRIN populations resulted
in a total of 24 significant marker-trait associations, representing 23 QDRL as defined by hap-
lotype blocks. These 23 QDRL were comprised of 12 QDRL identified from the C2-SK popula-
tion and distributed to ten chromosomes, three QDRL on Chr13 identified in the C2-US
population, one QDRL on Chr13 identified in the C2-GRIN population, and seven QDRL
identified from the C2 set and distributed to five chromosomes (Table 3, Fig 2). Three quanti-
tative trait loci (QTL) associated with non-inoculated traits were identified from the GRIN
population and located on Chr02. None of the QTL coincided with the identified QDRL (S7
Table, S9 Fig).
GWA analyses in the OH set, as well as OH-SK, OH-US, and OH-GRIN populations
resulted in a total of 24 marker-trait associations, representing 21 QDRL as defined by haplo-
type blocks. These 21 QDRL were comprised of seven QDRL identified from the OH-SK pop-
ulation distributed to four chromosomes; three QDRL identified from the OH-US population
and distributed to three chromosomes; six QDRL identified in the OH-GRIN population dis-
tributed to four chromosomes; and five QDRL identified using the OH set distributed three

Table 3. Quantitative disease resistance loci (QDRL) mapped in the C2 set.

Namea Popb Traitc -log10(p) PVEd Markere Flanking positionsf
C2-02-1 C2-SK IRW 5.49 2.78 ss715582994 43367206–43440684
C2-03-1 C2-SK IRRS 7.02 6.88 ss715586961 786873–821859
C2-03-2 C2-SK ΔRW 5.48 7.03 ss715586992 821859–875151
C2-03-3 C2-SK IRRS 7.43 4.04 ss715585633 3691222–3895958
C2-04-1 C2-SK IRW 9.03 5.52 ss715589155 6514173–6682383
C2-05-1 C2-SK IRW 17.32 5.71 ss715591632 41780982–42090709
C2-08-1 C2 set IPH 6.1 2.79 ss715602597 5472166–5709053
C2-08-2 C2-SK IRRS 11.02 1.17 ss715602910 9877098–9898176
C2-08-3 C2 set IPH 6.78 3.16 ss715600593 20295654–20975559
C2-11-1 C2 set ΔRW 5.97 3.11 ss715609313 26075475–26169302
C2-13-1 C2-US ΔSW 6.5 9.19 ss715617255 13389672–13550863
C2-13-2 C2-GRIN IRRS 6.04 0.21 ss715616837 15952204–16017061
C2-13-3 C2-SK ΔRW 5.96 10.37 ss715615656 18315025–18531998
ΔSW 6.58 13.11 ss715615656
C2-13-4 C2-US IRRS 5.44 5.03 ss715614099 19599094–19614217
C2-13-5g C2 set IPH 5.51 2.07 ss715614543 28001686–28051574
C2-13-6h C2 set IRW 6.21 3.45 ss715614993 30502735–30618405
C2-13-7i C2-US IRW 6.82 11.2 ss715615007 30646059–30654291
C2-14-1 C2-SK IRW 7.84 4.72 ss715618005 2131853–2153133
C2-15-1 C2-SK IRRS 5.72 1.01 ss715621545 2952387–3182673
C2-16-1 C2-SK IRRS 17.2 7.52 ss715623885 27437538–27660360
C2-17-1 C2 set ΔRW 7.32 4.54 ss715626781 33515060–33574931
C2-17-2 C2-SK IRW 7.18 2.38 ss715627019 36398362–36411792
C2-18-1 C2 set IPH 5.51 2.21 ss715630573 347317–441123
a
, Name of the QDRL in which “C2” indicates where the trait was mapped in the assay with isolate C2.S1, followed by “-##”in which the two digit number represents the
chromosome to which the QDRL is located, and “-#” in which the number represents the order the QDRL are found on the chromosome.
b
, SK, South Korea; US, North Central US; GRIN, full diversity of germplasm collection.
c
d
, PVE, prevent variation explained.
e
, Marker identified as associated with trait in GWA analysis
f
, Basepair position flanking QDRL determined by linkage.
g
, QDRL within 500kb of OH-13-1.
h
, QDRL within 500kb of OH-13-2, OH-13-3, OH-13-4, OH-13-5.
i
, QDRL within 500kb of OH-13-4, OH-13-5.
chromosomes. GWA analyses for non-inoculated traits identified 13 QTL distributed on 12

chromosomes (S10 Fig, S7 Table). On Chr05, a QTL for NPH was coincident with QDRL OH-
5-1 for IPH, indicating that this locus may control plant architecture rather than a QDRL, per
se.
Comparisons of GWA analyses between the C2 and OH sets

GWA analyses in the C2 and OH sets and the populations derived from these sets resulted in
the identification of 44 QDRL on 16 chromosomes; with 23 QDRL distributed to 12 chromo-
somes identified in the C2 set and 21 QDRL distributed to 10 chromosomes identified in OH

Table 4. Quantitative disease resistance loci (QDRL) mapped in the OH set.

Namea Popb Traitc -log10(p) PVEd Markere Flanking positionsf
OH-02-1 OH set ΔSW 5.58 2.69 ss715582345 38935468–39002760
OH-02-2 OH-SK IRRS 5.95 6.36 ss715582359 39090899–39126047
OH-03-1 OH-SK IRW 8.36 4.76 ss715586915 6074620–6378977
ISW 5.46 4.67
OH-03-2 OH-SK IRRS 6.63 8.47 ss715586985 7872384–8252615
OH-03-3 OH-SK ΔSW 10.04 8.58 ss715586306 42506684–42517511
OH-05-1 OH-GRIN IPH 7.08 7.43 ss715591382 36972839–37035513
OH-06-1 OH-US IPH 5.48 7.79 ss715595238 50603738–50852296
IRRS 5.91 8.71 ss715595238
OH-10-1 OH-SK IRW 5.32 4.19 ss715605487 998272–1022198
OH-10-2 OH-SK ISW 6.13 4.45 ss715606258 33174697–33499135
OH-11-1 OH-GRIN IRW 6.61 8.88 ss715610747 4563815–4586936
OH-11-2 OH-GRIN ΔSW 5.47 12.1 ss715610923 5898743–5962759
OH-12-1 OH-SK ΔRW 5.94 10.73 ss715611695 1804514–1844191
OH-13-1g OH-GRIN IRRS 5.62 6.75 ss715614516 27874365–27896769
OH-13-2h OH set IRW 8.06 2.61 ss715614895 29971253–30065880
OH-13-3h OH-US IRW 5.87 8.02 ss715614914 30086805–30144416
OH-13-4h,i OH set IRRS 5.98 2.57 ss715614952 30291675–30301385
IRW 7.44 2.28 ss715614952
OH-13-5h,i OH set ΔRW 5.75 3.06 ss715615020 30667266–30700217
OH-18-1 OH-US ISW 4.83 7.19 ss715629906 2128180–2155661
OH-18-2 OH set IRRS 5.91 2.93 ss715632090 54737619–54774006
OH-20-1 OH-GRIN IPH 5.41 8.89 ss715636836 1724545–1897580
OH-20-2 OH-GRIN IPH 5.55 7.72 ss715638609 45279755–45458003
a
, Name of the QDRL in which “OH” that indicates where the trait was mapped in the assay with isolate OH.121, is followed by “-##”in which the two digit number
represents the chromosome to which the QDRL is located, and “-#” in which the number represents the order the QDRL are found on the chromosome.
b
, SK, South Korea; US, North Central US; GRIN, full diversity of germplasm collection.
c
d
, PVE, prevent variation explained.
e
, Marker identified as associated with trait in GWA analysis
f
, Basepair position flanking QDRL determined by linkage.
g
, QDRL within 500kb of C2-13-5.
h
i
set. Little overlap was observed between the results of the OH set and C2 set. Only QDRL C13-
5, C13-6, C13-7, O13-4, and O13-5 (Tables 3 and 4; Figs 2 and 3) were identified in similar
regions (<500 kb apart) between OH and C2 sets.
Within a given set (C2 or OH), different QDRL were detected depending on the popula-
tion. For example, the C2-SK population identified different QDRL than the C2-US and
C2-GRIN. Similar differences were also observed among the QDRL identified from the popu-
lations within the OH set. In this case, the populations within a given set are structured and
allele frequencies are expected to contribute to the differences in the QDRL that were detected.
Indeed, there were significant differences in allele frequencies between populations (P
value < 0.01) at all identified QDRL (Fig 4).

Fig 2. Manhattan plot of genome wide association of layer test traits from the C2 set. Marker associations for the full C2 set and the C2-GRIN,
C2-SK, and C2-US populations for each trait are overlaid. For significant associations, markers for each population and trait are differentiated by the
shape and color of the marker, respectively. Significance thresholds were calculated for each population using SimpleM, only the highest (C2 set) and
lowest (C2-US) thresholds are displayed. Red shading of non-significant markers indicates the genetic regions associated with known Rps-genes.
Genomic positions previously identified significantly associated with quantitative resistance to P. sojae in previous GWA analyses are indicated by the
black vertical bars. Grey highlighted regions represent meta-QDRL, with the darker grey highlight indicating QDRL identified in exotic germplasm,
and the lighter grey representing QDRL identified from US cultivars.
There were also some differences in allele frequencies from the corresponding populations
within the C2 and OH sets (i.e., C2-US vs. OH-US, C2-SK vs. OH-SK and C2-GRIN vs.
OH-GRIN), despite sampling the same population-groups from the population structure anal-
ysis (Fig 1). While the majority of allele frequencies were very similar and not significantly dif-
ferent, eight alleles were significantly different between populations (Fig 5) with five
significant differences in allele frequencies between the US populations and three significant
differences between the C2 set and OH set. The ability to detect QDRL is affected by allele fre-
quency [79] and could contribute to the differences in identified QDRL.
Corroboration of the identified QDRL and direction of allelic effects

There was little overlap in the QDRL identified in the C2 set and OH sets. However, single
marker analyses, comparing the phenotypic means of PIs with the resistant or susceptible allele
at the identified QDRL, verified the direction and allelic effect in relevant traits (Figs 5 and 6).
At 43 of 44 QDRL, PIs with the resistance allele had BLUP values indicating a higher level of
resistance than PIs with a susceptible allele in both the population in which the QDRL was
identified and the corresponding population assayed with the alternative isolate (Figs 5 and 6).
The only exception was the significant marker for OH-11-2, which was not polymorphic in
the C2 set.

Fig 3. Manhattan plot of genome wide association of layer test traits from the OH set. Marker associations for the full OH set and the OH-GRIN,
OH-SK, and OH-US populations for each trait are overlaid. For significant associations, markers for each population and trait are differentiated by the
shape and color of the marker, respectively. Significance thresholds were calculated for each population using SimpleM, only the highest (OH set) and
lowest (OH-US) thresholds are displayed. Genomic locations of Rps-genes, regions identified as significantly associated with quantitative resistance to
P. sojae in previous GWA analyses, and meta-QDRL are indicated as described for Fig 3.
The QDRL markers were often significantly associated with QDR towards either OH.121
or C2.S1 in their respective populations. Using single marker analyses, most markers (37 of
44) had a significant marker effect for the isolate, population, and trait in which the association
was originally identified by GWA analysis. More surprising is that a large number (26 of 44) of
the single marker tests also had significant effects for QDR to the alternate isolate in the corre-
sponding population. Thus, in addition to the direction of allelic effects being the same
between the two isolates and their corresponding populations, these allelic effects were often
significant based on single marker analyses.
For most of the corresponding populations, the significant markers had a similar allele fre-
quency in both populations (e.g. C2-GRIN and OH-GRIN) (Figs 5 and 6). However, for nine
QDRL, the allele frequency was significantly different between populations or monomorphic
in one population. Of the 18 QDRL that were not confirmed in the relevant population for
resistance to the alternate isolate, one was monomorphic and three had significantly different
allele frequencies in the relevant population corresponding to the alternative isolate. In these
instances, differences in the marker effect may be due to population sampling rather than iso-
late or environment.
In both the C2 and OH sets, there was a significant correlation (P value <0.001) between
the number of resistant alleles and an overall resistance rank (Fig 7a and 7c). In the C2 set, the
most resistant quintile averaged 25.2 resistance alleles (of 44), significantly higher than the
other quantiles (Fig 7b). The OH set displayed a similar trend with the most resistant quintile

Fig 4. Allele frequencies of SNPs in the combined US, SK, and GRIN populations in the C2 and OH sets of PIs.
The frequency of SNP alleles was calculated by adding the total number of individuals with the first allele (first in
alphabetical order of SNP) divided by the total number of individuals in the population. The US population is
represented by grey dots, the SK population by black dots and the GRIN population with red dots. A χ2 test was
completed to test for significant differences in allele frequencies. All allele frequencies were significantly different (P
value < 0.01).
averaging 25.8 resistance alleles, significantly more resistance alleles than the other quintiles
(Fig 7d).
Meta-QDRL analysis
Comparison to previously identified QDRL would provide further evidence to validate our
findings as well as distinguish the novel QDRL identified in this study. While over a hundred
QDRL for resistance to P. sojae have been previously identified from bi-parental mapping
studies, many were identified in only a single study. Thus, in order to focus on the robust
QDRL, we completed a meta-analysis of QDRL.
In addition to four GWA studies (S8 Table), 16 biparental mapping studies have also been
completed (S2 Table), identifying 143 QDRL positions (77 positions on Soybase) [61]. The
studies were filtered such that only a single mapping study from the same biparental cross and
generation is represented and studies prior to 2010 were removed. The remaining 11 studies
describe 114 QDRL and were included in this meta-analysis. The meta-analysis of the QDRL

Fig 5. Allele frequencies and average BLUP values of PIs with resistant and susceptible alleles at QDRL identified
in GWA analyses in the US, GRIN, and set populations. The frequency of the resistant allele is indicated in the
“Freq” column. Significantly different allele frequencies (χ2 test, α = 0.05) and instances where one population is
monomorphic were indicated with grey shading. For resistant (red) and susceptible (black) alleles of each QDRL,
means and standard errors were calculated from the population in which the significant association was identified and
are indicated with points and bars, respectively. P values from t-test are presented in the “Pheno” column with P
value < 0.05 indicated with � , < 0.01 indicated with �� and < 0.001 indicated with �� . In order to consistently have
the resistance indicated as a higher BLUP value, BLUP values for inoculated root rot score (IRRS) were multiplied by
“-1”. Eight QDRL had a greater distribution of BLUP values and were displayed on a separate chart to aid readability.
(A) US population allele frequency and average BLUP values. (B) GRIN population allele frequency and average BLUP
values. (C) Set population allele frequency and BLUP values.
Fig 6. Allele frequencies and average BLUP values of PIs with resistant and susceptible alleles at the QDRL identified in GWA analyses in the
C2-SK and OH-SK populations. The frequency of the resistant allele is indicated in the “Freq” column, none were significantly different (χ2 test, α =
0.05). For resistant (red) and susceptible (black) alleles of each QDRL, means and standard errors were calculated from the population in which the
significant association was identified and is indicated with points and bars, respectively. P values from t-test are presented in the “Pheno” column with
P value < 0.05 indicated with � , < 0.01 indicated with �� and < 0.001 indicated with �� . In order to consistently have the resistance indicated as a
higher BLUP value, BLUP values for inoculated root rot score (IRRS) were multiplied by “-1”. Eight QDRL had a greater distribution of BLUP values
and were displayed on a separate chart to aid readability. Significantly different allele frequencies were identified with a χ2 test and are indicated with
grey shading.

Fig 7. Relationship between PI QDR level and the number of resistance alleles at the identified QDRL. The total
number of resistant alleles from all QDRL was calculated for all PI in the C2 set (a,b) and OH set (c,d). An overall
resistance score was calculated by ranking each PI on the basis of a summed rank of the PI for each inoculated trait.
The linear relationship between rank and resistant allele number was calculated in the C2 set (a) and OH set (c). The
average rank and resistant allele number was calculated in quintiles in the C2 set (b) and OH set (d). Different letters
indicate significant differences in rank between quintiles as determined by Fisher’s protected least significant
difference (P value < 0.05).
consolidated these results into 22 robust meta-QDRL distributed across ten chromosomes
(Table 5). Based on the reference genome (Wms.82.a2), the meta-QDRL are associated with an
average physical region spanning 5547kb (Table 5).
Colocalization between previous research and current results

We identified regions most often associated with resistance by comparing current results with
the four previous GWA analyses for QDR to P. sojae [55–59], as well as the meta-QDRL
(Table 5). QDRL C2-02-1, C2-03-3, C2-13-7, and C2-17-2 were < 500kb from regions identi-
fied in previous GWA analyses, adding evidence to these regions being important in QDR. No
such colocalizations were identified for QDRL from the OH set or populations (Table 6).
Five QDRL from our GWA analyses are near (within 500kb) or overlap with meta-QDRL;
m08-1 near C2-08-02 (C2-SK), C2-13-4 (C2-US) near m13-1, C2-15-1 (C2-SK) near m15-1,
C2-16-1 (C2-SK) near m16-2, and OH-18-1 (OH-US) near both m18-1 and m18-2 (Figs 2 and
3). Because specific criteria were used to determine which QDRL mapping studies to include
in the meta-analysis, a number of previously reported QDRL were not included. The QDRL
C2-04-1, C2-13-2, and C2-13-3 co-localized with previously reported “non”-meta-QDRL
because these QDRL were either not included in the meta-analysis or the QDRL were merged
into meta-QDRL at a slightly different genetic position (Table 6).
In addition to the 12 QDRL from our GWA analyses which overlapped with previously
identified QDRL, fourteen of the QDRL identified in the present study were within
or < 500kb from reported Rps-gene locations. QDRL C2-02-1 co-localized with RpsZS18. C2-
03-1 and C2-03-2 were near the Rps1 region. We identified QDRL C2-13-5, C2-13-6, and C2-
13-7 within the Rps3 region, and the two QDRL identified on Chr17, C2-17-1 and C2-17-2,
were within the Rps10 region. In the PIs phenotyped with OH.121, OH-18-2 was near Rps6;
OH-13-1, OH-13-2, OH-13-3, OH-13-4, and O13-5 were all near Rps3 on Chr13 (Table 6).

Table 5. Phytophthora sojae QDRL identified in meta-analyses of previously completed biparental mapping studies.
Namea Flanking Markersb Flanking Positionsc
m01-1 BARC-035199-07136—BARC_2.0_Gm01_3237203 1031156–3237203
m01-2 BARC_2.0_Gm01_3342559—Sat_414 3342559–52336556
m02-1 BARC-013499-00502—BARC-063263-18286 2411666–4581169
m02-2 BARC-047945-10443—BARC-043983-08572 15054401–15694465
m04-1 BARC-030751-06938—BARC-019015-03051 2062813–5241170
m06-1 BARC-05997-16280—ss1235978364 5449370–7564115
m06-2 BARC-053603-11920—Sat316 47944572–48016485
m06-3 BARC-063259-18282—ss1235977679 11363772–11755372
m08-1 BARC-032503-08989—BARC-057257-14650 7780999–9484485
m08-2 BARC-017983-02492—BARC-040893-07862 10473841–11762085
m08-3 BARC-059367-15768—BARC-050015-09290 40735744–42086643
m09-1 BARC-064511-18708—Satt417 11755372–18102265
m13-1 Sat_297—Sat_298 19951364–26976064
m13-2 BARC-018521-02928—Satt335 31794058–31831950
m13-3 AW756935—BARC-016585-02149 41606169–44344337
m15-1 Satt411—BARC-008231-00112 2517404–3964389
m15-2 BARC_2.0_Gm15_6841404—BARC_2.0_Gm15_9983279 6841404–9983279
m16-1 BARC_2.0_Gm16_298020—BARC_2.0_Gm16_759723 298020–759723
m16-2 BARC_2.0_Gm16_27864182—BARC_2.0_Gm16_58887013 58887013–27864182
m18-1 BARC-020839-03962—ss1235985359 981868–2396395
m18-2 Sat_141—BARC-005806-00273 2418920–3276029
m18-3 BARC_2.0_Gm18_5151897—Satt325 5151897–8587948
a
, The name of the meta-QTL, with the m prefix indicating that it is a meta-QDRL, followed by “-##”in which the two digit number represents the chromosome to
which the QDRL is located, and “-#” in which the number represents the order the QDRL are found on the chromosome.
b
, Left and right flanking markers determined by meta-QDRL analyses
c
, basepair position of flanking markers QDRL
Some QDRL identified in this study were novel in their association towards P. sojae disease
resistance, but were in regions previously associated with resistance to other soybean patho-
gens and pests. QDRL OH-03-3 is in a region associated with resistance to whiteflies and soy-
bean cyst nematode (SCN) [80]. QDRL OH-05-1 is in a region associated with resistance to
the toxins produced by Fusarium virguliforme [81,82]. QDRL OH-06-1 is near the position of
a QDRL associated with resistance to sudden death syndrome (SDS) [83–85]. QDRL C2-08-3
was within regions associated with Sclerotinia and SDS resistance [86,87]. QDRL C2-11-1 is
near QDRL for SCN [88]. QDRL OH-20-1 is in a region associated with QDR to SDS [82].
OH-20-2 in a region associated with QDR to SCN [80] (Table 6). Finally, the remaining 14
QDRL are in regions not previously associated with resistance to fungal, viral, or nematode
pathogens based on information available in Soybase [61] (March 1st, 2019).
Discussion
A large and diverse population of soybean accessions led to the
identification of 44 QDRL
In this study, six traits for QDR were measured for PIs sampled from the USDA collection,
representing germplasm from the US, the Republic of Korea, as well as PIs that were sampled
without geographic consideration. PIs were phenotyped following inoculation with either P.

Table 6. Summary of colocalizations of identified QDRL with previously identified QDRL, Rps-genes, or other pathogens.
QDRL Other QDRL Rps-genes Other Pathogensa Citation(s)
b
C2-02-1 Satt634 RpsZS18 [57]/S1 Table
C2-03-1 Rps1 S1 Table
C2-03-3 ss715585728 [55]
OH-03-3 SCN [80]
C2-04-1 14–7c S2 Table
OH-05-1 SDS [81,82]
OH-06-1 SDS [83–85]
C2-08-2 m08-1 Table 5
C2-08-3 Sclerotinia & SDS [86,87]
C2-11-1 SCN [82]
c
C2-13-2 1–1, 2–1, 4–1, 3–1, 9–2 S2 Table
C2-13-3 1–1, 2–1, 4–1, 3–1, 9–2c S2 Table
C2-13-4 m13-1 Table 5
C2-13-7 ss715615031b Rps3 [55]
OH-13-1 Rps3 S1 Table
C2-15-1 m15-1 Table 5
C2-16-1 m16-2 Table 5
C2-17-2 Satt301b Rps10 [57]/S1 Table
OH-18-1 m18-1 Table 5
OH-20-1 SDS [82]
OH-20-2 SCN [80]
a
, identified resistance loci to other pathogens when no P. sojae resistance colocalized with identified QDRL. Colocalization with other pathosystems was not searched if
colocalization was identified with other P. sojae resistance.
b
, Significant marker referenced in GWA analysis.
c
, Name of QDRL as stored in Soybase.
sojae isolate C2.S1 or OH.121, forming the two sets of 495 PIs in the C2 set and 478 PIs in the
OH set, respectively. Identification and comparison of QDRL for multiple isolates of P. sojae
can be informative for making breeding decisions, as it is genetically complex [89], likely con-
tributing to not only diverse virulence patterns but also other aspects of pathogenesis. Though
it would have been interesting to phenotype all PIs with both isolates, a limited number of
seeds were available from the USDA collection.
GWA analyses were completed separately depending on the aforementioned experimental
design, and though the traits were significantly correlated, unique QDRL were identified for
each measurement. Though correlated, it remains pertinent to evaluate multiple traits, as each
QDRL could be functioning though different molecular mechanism or different tissue and
may only be detected by measuring a specific trait [90]. Overall, 44 QDRL were identified, and

though GWA analyses are expected to identify large-effect and common alleles [91], here we
identified 44 small-effect QDRL explaining less than 14% of the variation, consistent with
results from previous mapping studies (S2 Table).
Superficially, the GWA analyses here appear to have identified many more QDRL than pre-
vious studies. For example, GWA analyses for QDR towards P. sojae have been completed in
more specific samples of germplasm and individually have identified a maximum of 16 mark-
ers associated with resistance [55]. Biparental mapping studies have also identified similar
numbers of QDRL [42–48]. For example, a cross of Conrad x Hefeng 25 identified eight
QDRL [44]; the cross of Conrad x Sloan identified 10 QDRL [48]; and crosses of PI 3998841 or
PI 407861A with OX20-8, identified 3 and 7 QDRL, respectively [51–53]. There were a total of
16 genetic regions associated with QDR to P. sojae identified using six nested populations in a
joint linkage mapping study with an average of 1.5 QDRL identified in individual populations
and 6 additional QDRL detected with joint linkage analyses [53].
However, both the diversity of PIs and the completion of analyses in multiple populations
likely contributed to the large number QDRL identified. Though the total number of QDRL
exceeded previous GWA results, analyses within individual population are comparable to pre-
vious research. For example, 11 QDRL were identified in the C2-SK population, and seven
QDRL in the OH-SK population. These results are similar to the seven QDRL identified in the
study completed in SK germplasm by Schneider et al. (2016) [55]. GWA analyses in Chinese
breeding lines have resulted in identifying three or one QDRLs [57,58], likewise the C2-US
and OH-US populations, consisting primarily of US cultivars, resulted in the identification of
3 QDRL.
The high level of soybean diversity in the full C2 set or OH set, may allow for the detec-
tion of novel QDRL, but it also increases the population structure, and, therefore, the risk of
false positives [92]. However, efforts to reduce false positives in GWA analyses were applied
by using models that incorporated population structure and kinship among PIs [93]. The
false-positive rate was also increased because multiple GWA analyses were completed for
each population within each set (C2 and OH) of PIs. The methodology in this study was
similar to that of Bandillo et al. (2015) [94], where a broad collection of PIs was initially
sampled for GWA analysis, followed by analyses of four subsets from the initial collection to
identify QTL for seed protein and oil content. Though testing subsets of PIs in addition to
the full set does increase the false positive rate, we applied appropriate and stringent signifi-
cance thresholds specific to each population based on the respective effective marker num-
bers [71].
Many QDRL co-localize with previously identified loci for P. sojae

resistance, as well as represent novel loci for this pathosystem
Prior to this study, 21 biparental or GWA mapping studies for QDR towards P. sojae have
been completed (S2 Table). Additionally more than 30 Rps-genes/alleles have been identified
for race-specific resistance (S1 Table). Twenty-one of the QDRL reported in this study were
novel loci for this pathosystem, adding significantly to our knowledge of the genetic architec-
ture of QDR to P. sojae. The remaining 23 QDRL colocalize with previously identified QDRL
and/or Rps-genes. Depending on whether these co-localized resistances are in cis, in trans, or
controlled by the same gene(s), the colocalization of these QDRL could have significant breed-
ing implications.
Colocalization was identified by comparing the QDRL identified here with the 143 QDRL
reported from 16 biparental mapping studies positions (S2 Table). Though the expression of
QDR towards P. sojae can be dependent on environmental, methodological, and assay

conditions [47], in this study we consolidated these QDRL into 22 robust meta-QDRL distrib-
uted across 10 chromosomes. In total, twelve QDRL, or 27%, colocalized with previously iden-
tified loci from GWA analyses, non-meta-QDRL for P. sojae resistance or meta-QDRL. This is
similar to previous GWA studies where approximately 33% of QDRL co-localized with results
from other studies [55,57,59]. Five of the 22 meta-QDRL colocalized with QDRL we identified
in this study, strongly supporting the involvement of these regions in QDR to P. sojae. Though
only 5 QDRL colocalized with meta-QDRL, this analysis greatly facilitated the consolidation of
the growing number of identified QDRL for resistance towards P. sojae.
Rps-gene regions often overlap with QDRL regions [53,55]. In this study, 14 QDRL over-
lapped with or were within 500kb of previously reported Rps-gene regions. Though pheno-
typically distinct, the colocalization of Rps-genes is conspicuous, coaxing the research
question: Are canonical R-genes functioning in a QDR? Indeed, researchers have previously
proposed that QDR may be an R-gene-mediated response that does not confer complete
resistance, resulting in race-specific QDRL [95]. While studies have previously identified
QDRL that have been significant for one isolate and not other isolates [48], at this time, no
significant race or isolate × QDRL interaction has been reported, which would be the hall-
mark of a bona fide race-specific QDRL. Few studies on QDR to P. sojae have specifically
addressed race-specificity of QDRL, and those that have tested for race-specific QDRL did
not identify any significant isolate × QDRL interaction [96]. In this study, isolate, popula-
tion, as well as environment, were all confounded. Thus, we were unable to test the race or
isolate-specificity of the QDRL.
There was little overlap in QDRL identified between the C2 set and OH set
The C2 set and OH set were both comprised of US, SK and GRIN populations sampled from
the USDA germplasm collection. Though the germplasm was similar, the two sets varied for
isolate and the greenhouse environment, and while similar numbers of QDRL were identified
in both the C2 set and OH set, there was little overlap between the QDRL identified with each
isolate, among populations, or for each trait. With the exception of a region on Chr13, the
QDRL identified in either the C2 set or OH set were unique to the set.
As in other host-pathosystems, QDR is often a complex trait [28]. This type of genetic com-
plexity can be observed in the foliar disease of maize, Southern Corn Leaf Blight, where GWA
analysis for resistance in the maize NAM population resulted in the identification of 32 QDRL
[97]. Similarly, over 100 QDRL have been identified for the well-studied Fusarium head blight
resistance in wheat [98]. Based on current and previous mapping studies for resistance to P.
sojae, we can estimate that greater than 60 loci are contributing to QDR. The majority of iden-
tified QDRL toward P. sojae in soybean explain less than 15% of the variation in the population
in which they were mapped. The small individual effect of each allele contributes to the com-
plexity of the resistance phenotype itself, as well as to the genetic analysis of the trait. In fact,
while many QDRL have been reported as isolate-specific or specific to an individual resistance
trait or assay [47,48,53,55], it is unclear whether these specific QDRL are due to the marginal
significance of small effect loci, or the functional complexity of the QDR. Interestingly, though
QDRL were only identified with one isolate of P. sojae, there were significant differences in the
response trait between the resistant and susceptible alleles both within the populations in
which the QDRL were identified as well as within the corresponding population assayed with
the alternate isolate. This provides independent corroboration for these QDRL and indicates
that many, if not all, of the 44 QDRL are valid.
Among the populations that made up each isolate set, we observed unique QDRL, with no
overlap in the genetic positions of QDRL identified among populations within each isolate set.

As expected, we also observed unique alleles and genetic structure for each population within
an isolate set. Populations were sampled from specific groups, which were determined by a
population structure analysis, and possessed differences in the number of segregating markers
as well as significantly different allele frequencies at QDRL. Thus, the lack of overlap between
QDRL identified in each population is likely a result of some combination of differences in
resistance allele polymorphism, allele frequency, and/or LD.
Targeting multiple QDRL for different traits may provide improvement of

QDR in elite cultivars
QDR towards P. sojae is thought to consist of multiple mechanisms that interact to produce a
defense response [99]; as such, combining QDRL involved with different aspects of resistance
could produce the highest levels of resistance. However, the question remains: How applicable
are individual minor effect QDRLs, possibly only contributing to one isolate or trait, for the
improvement of elite cultivars? Indeed, these are small effect QDRL, however one of the pro-
posed benefits of QDR is durability, and minor effect QDRL are predicted to be more durable
than large-effect QDRL with wide-spread deployment [91].
Specifically for US germplasm, there has been an improvement in QDR over time, but this
began to plateau in the 1980s [100]. Indeed, US populations in this study had lower levels of
QDR than the SK and GRIN populations. In the C2 set, only 10 of the top 50 most resistant PIs
are from the C2-US population. The OH set is similar, with 11 US PIs in the top 50 most resis-
tant PIs.
Among the most resistant PIs from the C2-US population are four Clark isolines
(PI547538, PI547583, PI547476, PI547528) and one Williams isoline (PI591509) [101]. Both
Clark and Williams have been reported to have moderate to high levels of QDR against P.
sojae [55]. Likewise in the OH set, the most resistant lines were also from Clark (PI547496)
and Williams (PI547852, PI547496, and PI634758) derivatives [105,106]. In addition, among
the 11 resistant US PIs were cultivars OH FG2 (PI584470), Lonoke (P633609), and Athow
(PI595926), all of which had been previously reported as moderate to highly resistant [38, 102–
105]. Thus, disease assays in each of the US populations have repeatedly identified known and
often well utilized sources of partial resistance to P. sojae. At a minimum, Clark and Williams
have been widely incorporated into breeding programs [106]; therefore, it is possible many of
the QDR alleles identified in the US populations have already been deployed in US
germplasm.
Overall, there was a correlation between the number of resistant alleles and the overall resis-
tance levels, indicating the value of stacking QDR alleles. In other pathosystems, such as rice-
Magnaporthe oryzae, pyramiding of QDRL increased resistance [107]. Similarly, pyramiding
QDR alleles for resistance to Fusarium head blight in wheat increased resistance. Interestingly,
among the top 50 most resistant PIs, PIs from the US populations averaged fewer QDR alleles
than the PIs from the GRIN and SK populations. Thus, there may be room for continued
improvement by incorporating additional QDR alleles.
Based on this study, valuable and previously untested QDR alleles are available to soybean
breeders to increase the diversity in their genetic base as they attempt to improve elite cultivars,
notably from PIs originating from SK, which had high levels of QDR in this study and previous
research [55,56]. The complex genetic architecture of QDR towards P. sojae indicates the
importance of stacking multiple QDR alleles or a potential role for tools such as genomic selec-
tion. Efforts to introduce alleles contributing to a number of QDR traits are expected to pro-
vide the most durable and highest levels of QDR.

Supporting information
S1 Table. Positions of Rps-genes/alleles in the soybean genome.
(XLSX)
S2 Table. Published quantitative disease resistance towards Phytophthora sojae mapping
studies.
(XLSX)
S3 Table. Overall ranking of resistance for plant introductions in the C2 set. PIs were
ranked from most resistance to least resistant for the four inoculated traits, ΔCRW and ΔCSW,
ranks were summed and re-ranked.
(XLSX)
S4 Table. Overall ranking of resistance for plant introductions in the OH set. PIs were
ranked from most resistance to least resistant for the four inoculated traits, ΔCRW and ΔCSW,
ranks were summed and re-ranked.
(XLSX)
S5 Table. FastStructure population number selection.
(XLSX)
S6 Table. Genetic variance and heritability in each population.
(XLSX)
S7 Table. Significant marker-trait associations identified for non-inoculated traits.
(XLSX)
S8 Table. Results of previously completed GWA analyses for P. sojae quantitative disease
resistance.
(XLSX)
S1 Fig. Comparison of QQ-plots of different genome-wide association analysis models
implemented in GAPIT. Models represented (1) GLM; general linear model, (2) MLM;
mixed linear model, (3) CMLM; compressed mixed linear model, (4) MLMM; multiple linear
mixed model.
(TIFF)
S2 Fig. Population clustering using fastStructure indicated nine subpopulations within the
SK populations. (A) Nine subpopulations identified in the C2-SK PIs. (B) Nine subpopula-
tions identified in the OH-SK PIs (C) Model complexities from one to ten were tested identify-
ing a plateauing marginal likelihood value at k = 9.
(TIFF)
S3 Fig. Population clustering using fastStructure indicated six subpopulations within the
US populations. (A) Six subpopulations identified in the C2-US population. (B) Six subpopu-
lations identified in the OH-US population. (C) Model complexities from one to eight were
tested identifying a plateauing marginal likelihood at k = 6.
(TIFF)
S4 Fig. Linkage disequilibrium decay in heterochromatic and euchromatic regions and sig-
nificance thresholds calculated using Meff. Linkage between markers (r2) as a function of
physical distance (base pair) calculated in the 974 plant introductions used in GWA analyses.
Significance thresholds include: a, effective marker number; b, calculated significance threshold
[-log10(0.05/effective marker number]; c, estimated effective marker density in markers per

kilobases.
(TIFF)
S5 Fig. Histogram distribution and qq-normality plots of the 495 PIs in C2 set used for
GWA mapping. IRRS, inoculated root rot score; IRW, inoculated root weight; ISW, inocu-
lated shoot weight; IPH, inoculated plant height; ΔRW, change in root weight; ΔSW, change in
shoot weight; NRW, non-inoculated root weight; NSW, non-inoculated shoot weight; NPH,
non-inoculated plant height.
(TIFF)
S6 Fig. Comparison of BLUP values between the US, SK, and GRIN populations in the C2
set. The GRIN population (red) the SK population (white) boxplots, and the US population
(grey) populations are represented for all nine traits: IRRS, inoculated root rot score; IRW,
inoculated root weight; ISW, inoculated shoot weight; IPH, inoculated plant height; ΔRW,
change in root weight; ΔSW, change in shoot weight; NRW, non-inoculated root weight; NSW,
non-inoculated shoot weight; NPH, non-inoculated plant height. Significantly differences in
population determined using Fisher’s protected LSDs (P value <0.05) are indicated with letters.
(TIFF)
S7 Fig. Histogram distribution and qq-normality plots of the 478 PIs in OH set used for
GWA mapping. IRRS, inoculated root rot score; IRW, inoculated root weight; ISW, inocu-
lated shoot weight; IPH, inoculated plant height; ΔRW, change in root weight; ΔSW, change in
shoot weight; NRW, non-inoculated root weight; NSW, non-inoculated shoot weight; NPH,
non-inoculated plant height.
(TIFF)
S8 Fig. Comparison of BLUP values between the US, SK, and GRIN populations in the OH
Set. The GRIN population (red) the SK population (white) boxplots, and the US population
(grey) populations are represented for all nine traits: IRRS, inoculated root rot score; IRW,
inoculated root weight; ISW, inoculated shoot weight; IPH, inoculated plant height; ΔRW,
change in root weight; ΔSW, change in shoot weight; NRW, non-inoculated root weight;
NSW, non-inoculated shoot weight; NPH, non-inoculated plant height. Significantly differ-
ences in population were tested using Fisher’s protected LSDs (P value <0.05) are indicated
with letters.
(TIFF)
S9 Fig. Manhattan plot results of non-inoculated traits in the C2 set. Population the
marker-trait association was identified in via the shape of the significant marker. The specific
trait is identified by the color of the marker. Significance thresholds calculated using SimpleM
are displayed for the C2 set, and the C2-US Population.
(TIFF)
S10 Fig. Manhattan plot results of non-inoculated traits in the OH set. The figure shows
which population the marker-trait association was identified in via the shape of the significant
marker. The specific trait is identified by the color of the marker. Significance thresholds calcu-
lated using SimpleM are displayed for the OH set, and the OH-US Population.
(TIFF)
Acknowledgments
The authors thank Dr. Colin Davis, Dr. Stephanie Karhoff, Dr. Kyujung Van, Aliya Ansari,
Lily Thompson, Emma Kline, Brandon Bowers, and Hunter Jarosz for technical assistance in

greenhouse assays. The authors also thank the Ornamental Plant Germplasm Center for their
generous provision of greenhouse space.
Author Contributions
Conceptualization: William Rolling, Leah K. McHale.
Investigation: William Rolling, Rhiannon Lake.
Writing – original draft: William Rolling, Anne E. Dorrance, Leah K. McHale.
Writing – review & editing: William Rolling, Rhiannon Lake, Anne E. Dorrance, Leah K.
McHale.
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Genome-Wide Association Analyses of Quantitative Disease Resistance in Diverse Sets of Soybean (Glycine Max (L.) Merr.) Plant Introductions

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Genome-Wide Association Analyses of Quantitative Disease Resistance in Diverse Sets of Soybean (Glycine Max (L.) Merr.) Plant Introductions

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RESEARCH ARTICLE

Genome-wide association analyses of

Funding: This work was supported in part by an

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Materials and methods

Germplasm diversity and selection

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Quantitative disease resistance assays

Phytophthora isolates and Rps-gene assays

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estimations increasing with model complexities (number of population-groups) from 1 to 20

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Quantitative disease resistance of PIs in the C2 set

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Quantitative disease resistance of PIs in the OH set

All displayed correlations were significant at a P value of < 0.001

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Genome-wide association analyses

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Table 3. Quantitative disease resistance loci (QDRL) mapped in the C2 set.

chromosomes. GWA analyses for non-inoculated traits identified 13 QTL distributed on 12

Comparisons of GWA analyses between the C2 and OH sets

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Table 4. Quantitative disease resistance loci (QDRL) mapped in the OH set.

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Corroboration of the identified QDRL and direction of allelic effects

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Colocalization between previous research and current results

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Many QDRL co-localize with previously identified loci for P. sojae

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Targeting multiple QDRL for different traits may provide improvement of

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environments. Scientific reports. 2017; 7: 3554. https://doi.org/10.1038/s41598-017-03695-9 PMID:

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