Articles

Genome-wide association study using whole-genome

sequencing rapidly identifies new genes influencing
agronomic traits in rice
Kenji Yano1, Eiji Yamamoto2, Koichiro Aya1, Hideyuki Takeuchi1, Pei-ching Lo1, Li Hu1, Masanori Yamasaki3,
Shinya Yoshida4, Hidemi Kitano1, Ko Hirano1 & Makoto Matsuoka1
© 2016 Nature America, Inc. All rights reserved.

A genome-wide association study (GWAS) can be a powerful tool for the identification of genes associated with agronomic traits
in crop species, but it is often hindered by population structure and the large extent of linkage disequilibrium. In this study, we
identified agronomically important genes in rice using GWAS based on whole-genome sequencing, followed by the screening of
candidate genes based on the estimated effect of nucleotide polymorphisms. Using this approach, we identified four new genes
associated with agronomic traits. Some genes were undetectable by standard SNP analysis, but we detected them using gene-
based association analysis. This study provides fundamental insights relevant to the rapid identification of genes associated with
agronomic traits using GWAS and will accelerate future efforts aimed at crop improvement.

Crop improvement through genetics and breeding is essential for
increasing yields and feeding a growing world population1. The iden-
tification and characterization of genes associated with agronomi-
cally important traits is indispensable for both understanding the
genetic basis of phenotypic variation and efficient crop improvement.
Identification of genes underlying agronomic quantitative trait loci
(QTLs) is usually performed using biparental mapping populations,
such as F2 and recombinant inbred lines2,3. However, these strategies
have limitations such as low mapping resolution and limited genetic
diversity between the mapping population parents. For example, only
npg
represent strong population structure that generates spurious associa-
tions between the phenotype and unlinked markers. Although several
statistically robust models have been developed, it must be noted that
false positives arising from population structure in crops may not be
completely controlled4–6. To address this problem, a reconstruction
of population has been proposed, which is represented by a ‘nested
association mapping population’22–25 and a ‘multiparent advanced
generation intercrossing population’26–28. The second reason for the
difficulty in identifying unknown genes is the large extent of link-
age disequilibrium (LD). The extent of LD in the human genome is

two allelic variations are analyzed (one per parent) in a biparental
population, which means that various alleles occurring in other plants
are missed. A GWAS that analyzes associations between nucleotide
polymorphisms and phenotypic variance using a diverse population
set is a powerful tool for the identification of genes associated with
agronomic traits, because this strategy can be used to detect many
natural allelic variations simultaneously in a single study4–6. Recent
advances in high-throughput sequencing technologies have enabled
rapid and accurate resequencing of a large number of genomes7–15.
This is expected to revolutionize GWAS because the approach will
not only prevent ascertainment bias in studies with limited nucleotide
polymorphisms but also facilitate more direct searches for the causal
variant(s) underlying phenotypic diversity16,17.
GWAS of numerous crop species have detected many QTLs asso-
ciated with agronomic traits9,12,18–21. However, it is often difficult
to identify unknown genes associated with the QTLs owing to two
major reasons4–6. The first is that diversity panels of crop species often
generally smaller than gene size29, whereas LD in plants often ranges
over several hundred kilobases, especially in self-pollinating crops
such as rice and soybean30. This results in the inclusion of many
candidate genes in a single LD block exhibiting a significant signal,
thus entailing the need for additional experiments to conclusively
identify the causal gene(s).
In this study, we attempted to identify genes associated with agro-
nomic traits using GWAS with minimum additional experiments (i.e.,
only transgenic complementation tests). We focused on rice (Oryza
sativa L.), which feeds more than three billion people in the world31.
Furthermore, rice is a good candidate because of its extremely
strong population structure and the large extent of LD owing to self-
pollination19. To perform GWAS efficiently, we carefully selected a
population with low population structure but large phenotypic diver-
sity. By performing whole-genome sequencing, we refined the number
of candidate genes based on the estimated functional importance of
each nucleotide polymorphism. We demonstrated that with careful

1Bioscience and Biotechnology Center, Nagoya University, Nagoya, Japan. 2NARO Institute of Vegetable and Tea Science, Tsu, Japan. 3Food Resources Education and
Research Center, Graduate School of Agricultural Science, Kobe University, Kasai, Hyogo, Japan. 4Hyogo Prefectural Research Center for Agriculture, Forestry and
Fisheries, Kasai, Hyogo, Japan. Correspondence should be addressed to M.M. (makoto@agr.nagoya-u.ac.jp) and E.Y. (yame@affrc.go.jp).

Received 2 February; accepted 26 May; published online 20 June 2016; doi:10.1038/ng.3596

Nature Genetics  VOLUME 48 | NUMBER 8 | AUGUST 2016 927

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Table 1  Comparison of coefficient of variation for phenotypic
values between the 176 Japanese varieties used in this study and
the rice diversity panel reported in ref. 19
Days to
n Year heading
Varieties tested 176 2013
2014
Rice diversity panel 413
experimental design, GWAS could be a powerful tool for the rapid
identification of genes associated with agronomic traits.
RESULTS
Characterization of a population used in this study
To perform GWAS efficiently, it is important to select a population
that is not genetically highly structured and interrelated yet exhibits
high phenotypic diversity. We focused on 176 japonica rice varieties
developed in breeding programs conducted in Japan (Supplementary
Table 1 and Supplementary Fig. 1) for the following two reasons.
First, according to the pedigree information on these varieties (the
© 2016 Nature America, Inc. All rights reserved.
rice cultivar database; see URLs), they are genetically interrelated to
each other, and we expected them not to represent a highly structured
population (see below). Second, although we selected the varieties
from restricted pedigrees, the phenotypic diversity of agronomic
traits was almost comparable to that of the global collection of diverse
rice reported previuosly19 (Table 1, Fig. 1a–d and Supplementary
Table 2). This suggested that the varieties contain various QTLs asso-
ciated with agronomic traits.
To obtain nucleotide polymorphism information, we performed
whole-genome sequencing of the 176 varieties, and obtained a total
of 383.8 Gb of sequence, with an average depth of 5.8× and coverage
of 91.2% of the reference genome32 (Supplementary Table 3).
After removing nucleotide polymorphisms with missing rates ≥ 0.25
and minor allele frequency < 0.05, we generated a final set of 426,337
SNPs and 67,544 insertion-deletions (indels). Among the nucleotide
polymorphisms, 43,323 SNPs induced nonsynonymous substitutions,
whereas 1,678 and 1,656 indels induced frameshift and non-frameshift
mutations, respectively (Supplementary Table 4).
npg
Using these nucleotide polymorphisms, we performed principal
component analysis (PCA) to quantify the population structure of these
176 varieties (Fig. 1e). The score plot of principal components showed
continuous distribution without any distinct clusters, indicating
a b
Number of plants
Number of plants
75 60
50 40
25 20
0 0
–25 0 25
Normalized time to
heading (d)
c 100 d 60
Number of plants
Number of plants
75
40
50
20
25
0 0
−10 −5 0 5 10
Normalized panicle
length (cm)
928 VOLUME 48 | NUMBER 8 | AUGUST 2016  Nature Genetics
that the varieties we selected did not represent a highly structured
population. The extent of LD is another important factor determining
the efficiency of GWAS4–6. The decay of LD with physical distance
Coefficient of variation between SNPs occurred at 445 kb (r2 = 0.2), which is comparable to
Plant Panicle Leaf that of a previous study that used a more genetically diverse popula-
height length blade width tion19 (Supplementary Fig. 2). This indicated that the use of these
11.93 19.42 10.98 15.66 varieties had no substantial disadvantage as compared to that of the
  9.27 15.28 11.43 12.58 other sets of japonica germplasm, with respect to LD. Nevertheless, the
11.86 18.09 14.51 20.16 extent of LD was still problematic because it resulted in the inclusion
of tens to hundreds of candidate genes within a LD block, a problem
that is addressed below.
Validation of rapid gene identification using GWAS
Using a linear mixed model with correction of kinship bias
(Supplementary Fig. 3), we performed GWAS for heading date (i.e.,
days to heading) to assess the potential of our GWAS design for causal
gene identification. Heading date is a suitable trait for this purpose
because many genes with natural allelic variations have been identi-
fied in previous studies33. Using the linear mixed model, we con-
sistently detected 26 loci exceeding a significant threshold (−log10
P ≥ 4.77) in both years of experiment performed in the present study
(Supplementary Table 5). We focused on five of these loci, includ-
ing the top three peaks located on chromosomes 1, 6 and 11, and
two peaks with positions correlating with known heading date (Hd)
genes Hd6 and Hd2 (Fig. 2a).
At the terminal end of the long arm of chromosome 3, there was
a peak that mapped close to Hd6 (ref. 34) (Fig. 2a). We estimated
a candidate region from 30.50 Mb to 31.76 Mb (1,266 kb) by using
pairwise LD correlations (r2 ≥ 0.6) (Supplementary Fig. 4a). We clas-
sified all the polymorphisms in the candidate region into five groups.
Group I included polymorphisms that were significantly associated
with trait variation in the GWAS (−log10 P ≥ 4.77), and predicted
to induce amino acid exchange or to change splicing junctions (GT
or AG at the start or end of intron, respectively). Group II included
polymorphisms significantly associated with trait variation as group
I and were located at the 5′ flanking sequences of genes (≤2 kb from
the first ATG, for example, promoter region). Group III included
polymorphisms significantly associated with trait variation as group
I and located within a gene but did not meet the criteria for group
I or II (for example, located in a coding region but not predicted
to change an amino acid, an intron or a 3′ noncoding sequence).
Group IV included polymorphisms significantly associated with trait
variation as group I and located on outside coding regions. Group
V included polymorphisms not significantly
associated with trait variation. Although the
e candidate region on chromosome 3 con-
tained 297 polymorphisms, we assigned
0.15 Figure 1  Phenotypic diversity and genetic
structure of the Japanese rice varieties. (a–d)
0.10
Histograms of zero mean normalized phenotypic
0.05 values of days to heading (a), plant height (b),
PC2 (6.4%)
−50 0 50 100
Normalized plant panicle length (c) and leaf blade width (d).
height (cm) 0.00
Yellow and gray bars represent the 176
−0.05
Japanese rice varieties used in this study (data
from phenotyping performed in 2013) and the
−0.10
−0.15
diversity panel reported in ref. 19, respectively.
(e) PCA for the 176 Japanese rice varieties
−0.1 0 0.1 0.2 0.3 0.4 based on whole-genome sequence data. PC1
PC1 (8.5%) and PC2 indicate score of principal components
−0.5 0 0.5 1 and 2, respectively. Values in parentheses
Normalized leaf blade indicate percentage of variance in the data
width (cm) explained by each principal component.
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Figure 2  GWAS for days to heading and a 10 Hd1

c LOC_Os01g62780
identification of the causal gene for the peak Hd6 Chr. 1
Hd2
on chromosome 1. (a) Manhattan plot for 8
(+ strand)
days to heading. Dashed line represents the

–log10 (P)
+328
6
significance threshold (−log10 P = 4.77). Hap. A GTT (V)
Arrowheads indicate the position of strong 4 Hap. B ATT (I)
peaks that did not localize with the known Hd
2
genes investigated in this study. (b) Local d 2013 P = 4.20 × 10
–17
2014 P = 9.39 × 10
–18

Manhattan plot (top) and LD heatmap (bottom) 0 130

Chr. 1 2 3 4 5 6 7 8 9 10 11 12
surrounding the peak on chromosome 1. Arrow 110
indicates the position of nucleotide variation in b 10
120
100
LOC_Os01g62780. Dashed lines indicate the 8 110

Days to heading

Days to heading
candidate region for the peak. (c) Exon-intron

–log10 (P)
6 90
structure of LOC_Os01g62780 and DNA 100
polymorphism in that gene. (d) Boxplots for 4
90
days to heading based on the haplotypes (Hap.) 2 80
for LOC_Os01g62780 in 2013 (left) and 2014 0 80
(right). Box edges represent the 0.25 quantile Chr. 1 (Mb) 36.2 36.4 36.6 36.8 37.0 70
70
and 0.75 quantile with the median values shown
by bold lines. Whiskers extend to data no more than Hap. A B Hap. A B
(n = 109) (n = 67)
1.5 times the interquartile range, and remaining data are
indicated by dots. Differences between the haplotypes were e VEC Hap. A Hap. B f 120
**
analyzed by Welch’s t-test. (e) Image of transgenic plants transformed

Days to heading
110
with empty vector (VEC), haplotype A (Hap. A) and haplotype B (Hap. B).
© 2016 Nature America, Inc. All rights reserved.

n.s.
Red arrows indicate panicle exsertion. Scale bar, 15 cm. (f) Days 100
r2
to heading of the transgenic plants. Error bars, s.d. (n = 20).
**P < 0.01; n.s., not significant (Welch’s t-test). 0 0.2 0.4 0.6 0.8 1.0 90

80
only one polymorphism to group I (Supplementary Fig. 5), whereas

H .A
.B
VE
ap
ap
we assigned six polymorphisms to group II and three to group III.

The polymorphism in group I induced a premature stop codon in
the coding sequence of LOC_Os03g55389, which is identical to Hd6
(ref. 34) (Supplementary Fig. 4b). The varieties carrying haplotype
A (we refer the haplotype corresponding to reference genome as ‘A’
type and the other as ‘B’) showed earlier heading date than varieties
carrying haplotype B (Supplementary Fig. 4c), which is consistent
with the previous study on Hd6 (ref. 34).
Similarly, we analyzed a peak on chromosome 7, which mapped close
to Hd2 (ref. 35) (Supplementary Fig. 6a). The candidate region was
predicted to map from 29.35 to 29.70 Mb (346 kb), and contained 124
polymorphisms (Supplementary Fig. 7). Among these, there was only
one polymorphism assigned to group I, which was located on LOC_
npg
H
function of its rice homolog was unknown. The polymorphism on
LOC_Os01g62780 induced an amino acid exchange in the region
conserved among homologs in other plant species (Supplementary
Fig. 9). The residue of haplotype A (valine) is identical to that in
Arabidopsis HESO1, whereas the residue of haplotype B (isoleucine) is
identical to all other homologs compared in this analysis. O. rufipogon
(ancestral wild species of cultivated rice) and indica (another major
subspecies in cultivated rice) contained isoleucine at this position,
suggesting that haplotype B is the original haplotype in Oryza species
(Supplementary Fig. 9a). Varieties carrying haplotype B showed a
later heading date than haplotype A (Fig. 2d). To confirm the effect of
this gene on heading date, we introduced the entire genome sequence

Os07g49460 and was identical to Hd2 (ref. 35). The varieties contained
three haplotypes (Supplementary Fig. 6b). Varieties carrying haplotype C,
which is identical to the previously identified natural allelic variation
of Hd2 (ref. 35), showed an earlier heading date than those carrying
haplotype A or B (Supplementary Fig. 6c). This indicated that Hd2
is a gene responsible for this peak. The identification of Hd6 and Hd2
indicated that GWAS using whole-genome sequencing could be used to
efficiently identify genes associated with agronomic traits. We discuss
the second highest peak near Hd1 on chromosome 6 below.
Identification of new genes
We applied the same strategy to loci associated with heading date,
which have not been reported previously (i.e., chromosome 1 and 11)
(Fig. 2a). With respect to the locus on chromosome 1, the candidate
region was predicted to map from 36.30 to 36.65 Mb (346 kb) (Fig. 2b),
and included 91 polymorphisms (Supplementary Fig. 8). There were
eight group I polymorphisms, which were located within seven genes,
and all of these were annotated as transposon-related genes except
for LOC_Os01g62780, a homolog of Arabidopsis thaliana HEN1
suppressor 1 (ref. 36) (AT2G39740; HESO1) (Fig. 2c). Arabidopsis
HESO1 functions as a suppressor of the hen1 mutation and exhibits
pleiotropic phenotypes including delayed flowering37, whereas the
of haplotype A and B into Nipponbare carrying haplotype A. The
plants transformed with haplotype A showed no phenotypic change,
whereas plants transformed with haplotype B showed later heading
date when compared to the vector control and haplotype A plants
(Fig. 2e,f), which is consistent with the results in Figure 2d. These
observations clearly demonstrated that LOC_Os01g62780 is the causal
gene for the peak signal of days to heading on chromosome 1.
We then focused on the third highest peak on chromosome 11
(Fig. 2a). That locus exhibited pleiotropic associations with plant
height and panicle length (Fig. 3a,b). We estimated the candidate
region to be 4.33−4.79 Mb (463 kb) (Fig. 3c), and it contained 4,125
polymorphisms (Supplementary Fig. 10). We assigned 107 polymor-
phisms to group I, and these mapped to 24 genes. Most of these genes
(18 of 24) were annotated as either transposon-related, members of
the DnaK family or expressed protein, whereas the remaining six
were annotated as enzymes (five) or transcription factors (one). We
focused on LOC_Os11g08410, which is annotated as a GATA zinc
finger-type transcription factor. There were three haplotypes for
LOC_Os11g08410 (Fig. 3d). Haplotypes B and C contained one and
ten polymorphisms, respectively (Fig. 3d), whereas varieties carry-
ing haplotype C showed a later heading date, greater plant height and
larger panicle length than plants carrying haplotype A or haplotype B

Nature Genetics  VOLUME 48 | NUMBER 8 | AUGUST 2016 929

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Figure 3  GWAS for plant height and panicle a Plant height d LOC_Os11g08410
length, and identification of the causal gene for
15
the peak on chromosome 11. (a,b) Manhattan Chr.11

–log10 (P)
plots for plant height (a) and panicle length (b). 10
(– strand)
+590 +802 +809 +863 +916 +932 +935 +985 +994 +1,007 +1,286
Arrowheads indicate the position of strong TCA GTG CGC GTC GAC ACC ATC GGG
Hap. A
peaks investigated in this study. Dashed lines 5 (S) (V) (R) (V) (D) (T) (I) (G)
TCA GTG CGC ATC GAC ACC ATC GGG
represent significance thresholds (−log10 Hap. B
(S) (V) (R) (I) (D) (T) (I) (G)

P = 3.67 in a and −log10 P = 5.30 in b). 0

1 2 3 4 5 6 7 8 9 10 11 12 Hap. C 18 bp del
ACA GCG CAC GTC GCC
18 bp in
GCC
6 bp del
ACC GTG
(T) (A) (H) (V) (A) (A) (T) (V)
(c) Local Manhattan plot (top) and LD heatmap Chromosome
(bottom) surrounding the peak on chromosome 11. b Panicle length
e
Arrow indicates position of nucleotide 2013 2013 2013
8
variations in LOC_Os11g08410. Dashed lines 130 * 140 * n.s. *

–log10 (P)
6 n.s.

Panicle length (cm)

indicate the candidate region for the peak. 120
• •

Plant height (cm)

Days to heading
120 n.s. 25
(d) Exon structure of LOC_Os11g08410 and 4 110 •
•
•

DNA polymorphisms in that gene. Red- and 100

100
2 20
gray-shaded regions indicate nucleotide 90 80
variation significantly (−log10 P ≥ 4.77,
•
0 80 ••
•
1 2 3 4 5 6 7 8 9 10 11 12 60 15 •

3.67 and 5.30 for days to heading, plant Chromosome 70

•

c
•
height and panicle length, respectively) and
Plant height Hap. A B C Hap. A B C Hap. A B C
not significantly associated with phenotypic 15 (n = 27) (n = 106) (n = 43)
variation, respectively. in, insertion; del,
–log10 (P)

deletion. (e) Days to heading, plant height and 10 f g h 20

panicle length for indicated haplotypes for 90
5 110 * ** 18 **
80
LOC_Os11g08410. Data are presented as in
© 2016 Nature America, Inc. All rights reserved.

16

Panicle length (cm)

70 n.s.

Plant height (cm)

n.s. n.s.

Days to heading
Figure 2d. Differences between the haplotypes 106 14
0 60
Chr. 11 4.2 12
were statistically analyzed based on Tukey’s test 4.4 4.6 4.8 102 50
(Mb) 10
(*P < 0.05; n.s., not significant). (f–h) Days to 40 8
98 30
heading (f), plant height (g) and panicle length 6
20 4
(h) for transgenic plants transformed with empty 94
10 2
vector (VEC), haplotype A (Hap. A) and haplotype 90 0 0
C (Hap. C). Error bars, s.d. (n = 20). *P < 0.05,

.A

.C

C
.A

.C

.A
C

.C
VE

VE
VE
**P < 0.01; n.s., not significant (Welch’s t-test).

ap

ap

ap
ap

ap

ap
2
r

H
H

H
H

0 0.2 0.4 0.6 0.8 1.0
(Fig. 3e). Although the amino acid differ-
ences in haplotypes A and C were distributed
throughout the gene, it was difficult to predict the effect of these differ-
ences on function because they were located in nonconserved regions
(Supplementary Fig. 11). To examine whether this gene is causal for
the peak on chromosome 11, we introduced the genomic sequences of
haplotypes A or C into Nipponbare carrying haplotype A. The plants
transformed with haplotype C exhibited delayed heading, increased
plant height and larger panicle length (Fig. 3f–h and Supplementary
Fig. 12), which corresponds to the phenotypic variations presented
npg
H
into Nipponbare carrying haplotype A. The plants overexpressing
NAL1 from haplotype A showed decreased panicle numbers per plant
and increased leaf blade width relative to plants carrying the con-
trol vector, whereas plants transformed with haplotype B showed no
clear difference compared to controls (Fig. 4g–i and Supplementary
Fig. 15). Although NAL1 is known to be associated with several
agronomic traits, to our knowledge this is the first report to document
the effect of this gene on panicle number.

in Figure 3e. These results demonstrated that LOC_Os11g08410

is a causal gene for variations in these three agronomic traits and
demonstrated the utility of GWAS for identifying QTLs and genes
with pleiotropic effects.
The successful identification of genes associated with heading
date encouraged us to investigate genes associated with other agro-
nomic traits. On chromosome 4, we found a strong peak for panicle
number per plant (Fig. 4a), which overlapped with peaks for spikelet
number per panicle and leaf blade width (Fig. 4b,c), suggesting that
a single gene might have pleiotropic effects on these agronomically
important traits. We predicted the candidate region to be from
31.05 Mb to 31.41 Mb (358 kb), and it included 339 polymorphisms
(Fig. 4d and Supplementary Fig. 13). We assigned four polymor-
phisms to group I, and they mapped to four genes. Among these,
LOC_Os04g52479, which encodes NALLOW LEAF 1 (NAL1) (Fig. 4e
and Supplementary Fig. 14), has been previously reported to control
panicle size and flag leaf width, but its impact on panicle number is
less understood38,39. There were two haplotypes with distinct phe-
notypes: haplotype B produced more panicles per plant, smaller
spikelet numbers and narrower leaf blades than varieties containing
haplotype A (Fig. 4f). We introduced the cDNA sequence of NAL1
from haplotype A and B under the control of a constitutive promoter
Misleading association owing to allelic heterogeneity
Here we return to the second highest peak for days to heading on
chromosome 6 (Fig. 2a). The peak was located close to Hd1 (ref. 40),
which contributes to genetic diversity in heading date in the rice
breeding program41,42. Thus, we hypothesized that the causal
gene of the significant peak was Hd1. Using the approach described
above, we predicted the candidate region to be 7.81−8.38 Mb (564 kb)
(Fig. 5a). The candidate region did not include Hd1 (LOC_Os06g16370),
but Hd1 was localized in the next LD block (9.22–9.61 Mb),
which showed signals with lower −log10 P values than those in the
primary candidate region (Fig. 5a). We regarded this discrepancy as
an important issue for the efficacy of GWAS-based causal gene iden-
tification, which has been noted previously18,43,44; consequently, we
investigated its precise mechanism.
First, we surveyed why the polymorphisms in Hd1 were not sta-
tistically significant. In the population we used, there were eleven
haplotypes for Hd1 (haplotypes A–K), which included the null and
intermediate alleles previously reported 41 (Supplementary Figs. 16
and 17). In a standard GWAS, phenotypic distribution is independ-
ently compared in each nucleotide polymorphism site. Therefore, if
a gene includes allelic heterogeneity such as the case with Hd1, it

930 VOLUME 48 | NUMBER 8 | AUGUST 2016  Nature Genetics

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Figure 4  GWAS for panicle number per plant, a 7

Panicle number
per plant
e LOC_Os04g52479 (NAL1)
spikelet number per panicle and leaf blade 6

–log10 (P)
5 Chr. 4
width, and identification of the causal gene for 4
3 (+ strand)
the peak on chromosome 4. (a–c) Manhattan 2
plots for panicle number per plant (a), spikelet 1
+697
0
number per panicle (b) and leaf blade width (c). 1 2 3 4 5 6 7 8 9 10 11 12
Hap. A CAT (H)
Arrowheads indicate the position of strong Chromosome
peaks investigated in this study. Dashed lines b 15
Spikelet number
per panicle
Hap. B CGT (R)

represent significance threshold (−log 10 P =

–log10 (P)
5.39 in a, 4.60 in b and 5.50 in c). (d) Local 10
f 2013
–6
2013
–6
2013
–11
Manhattan plot (top) and LD heatmap (bottom) 5 P = 3.39 × 10 P = 6.89 × 10 P = 8.91 × 10
250 1.8

Spikelet number per panicle

surrounding the peak on chromosome 4.

Panicle number per plant

0 25

Leaf blade width (cm)

Arrow indicates the position of nucleotide 1 2 3 4 5 6 7 8 9 10 11 12
200 1.6
Chromosome
variation in LOC_Os04g52479. Dashed lines
indicate the candidate region for the peak.
c 10
Leaf blade width
20
150 1.4

–log10 (P)
(e) Exon-intron structure of LOC_Os04g52479 8 15
6 100 1.2
and DNA polymorphism in this gene. 10
4
(f) Panicle number per plant, spikelet number 2 1.0
50
per panicle and leaf blade width for the indicated 0 5
1 2 3 4 5 6 7 8 9 10 11 12
haplotypes of LOC_Os04g52479. Data are Hap. A B Hap. A B Hap. A B
Chromosome (n = 136) (n = 40)
presented as in Figure 2d. Differences between
the haplotypes were statistically analyzed d 6
Panicle number
per plant

based on Welch’s t-test. (g–i) Expression of 5 g h 14 i

–log10 (P)

1.2
LOC_Os04g52479 (NAL1) in the transgenic 4 14
© 2016 Nature America, Inc. All rights reserved.

** n.s. **

Panicle number per plant

plants (g). Panicle number per plant (h) and leaf 3 12 12 1.0

Leaf blade width (cm)

Expression of NAL1
blade width (i) for transgenic plants transformed 2
10 10
1 ** 0.8 n.s.
with empty vector (VEC), overexpression of 8 8 **
0 0.6
haplotype A (UBQ::Hap.A) and haplotype B Chr. 4 30.8 31.0 31.2 31.4 31.6 6
6
(UBQ::Hap.B). UBQ indicates maize ubiquitin (Mb) 0.4
4 4
promoter that was used for the overexpression of
2 2 0.2
LOC_Os04g52479. Error bars, s.d. (n = 12).
**P < 0.01; n.s., not significant (Welch’s t-test). 0 0 0

::H A
.B

::H C

::H .A
.B

::H C
::H .A
.B
.
BQ VE

BQ VE

BQ VE
BQ p
ap

BQ ap
ap

BQ ap
ap
U ::Ha
is difficult to achieve statistical significance
U

U
U
r2
because an allele-specific polymorphism is
compared with all other alleles, including 0 0.2 0.4 0.6 0.8 1.0

null, intermediate and fully functional alle-

les (Supplementary Fig. 16). Next, we surveyed why we observed
a strong peak in the LD block lacking Hd1. To illustrate the struc-
ture of this genome region schematically, we constructed a graphical
genotype in which major and minor polymorphisms in each site were
represented by blue and orange color (Online Methods) (Fig. 5b).
We classified the varieties into three groups based on Hd1 function,
npg
are under the significance threshold 48,49. On chromosome 6, some
genes in the 9.22–9.61 Mb region, which included Hd1 itself, formed
a higher peak than genes in the 7.81–8.38 Mb region (Fig. 5e). Thus,
we changed the candidate region and genes to the more plausible
LD block. In addition, we included Hd1 in top-ranking genes as the
other heading date genes identified in the present study (Fig. 5f

namely, haplotypes B and D (functional alleles), haplotype A (interme-
diate) and haplotypes E and F (null) (Fig. 5c), whereas in this analysis
we ignored other varieties containing minor haplotypes. The varie-
ties in each group showed a similar genome structure (Fig. 5b). This
comparison clearly indicated that the LD block including the highest
peak corresponds well to Hd1 function, i.e., functional (haplotypes
A, B and D) and nonfunctional (haplotypes E and F), whereas the LD
block including Hd1 showed no clear difference (Fig. 5b). Finally, we
confirmed the phenotypic distribution based on polymorphisms in
the highest peak (C or A at position 8267669 of chromosome 6) and
found that the average phenotypic value was clearly different between
polymorphism groups (Supplementary Fig. 17b). This represents a
theoretically anticipated situation that is designated synthetic asso-
ciation or indirect association45,46. When whole-genome sequence
data are available, another strategy for GWAS is to use a gene-based
association analysis that uses allelic differences as markers to analyze
the association between phenotypic variation and alleles47.
We assumed this strategy is effective to deal with the above-men-
tioned problem. Although no gene exceeded a stringent significant
threshold in the gene-based association analysis of heading date
(Fig. 5d,e and Supplementary Fig. 18), it has been reported that
top-ranking genes often include plausible candidates even if they
and Supplementary Table 6). These results indicate that gene-based
association analysis might be effective to deal with misleading asso-
ciations in single-polymorphism-based association analysis.
Owing to the result with Hd1, we resurveyed the phenotypes by
using gene-based association analysis and found that the strategy is
also efficient for identifying causal genes (Supplementary Fig. 19
and Supplementary Tables 6–12). Here we especially focused on awn
length. In the analysis of awn length by single-polymorphism-based
GWAS, we detected a strong peak (−log10 P = 24.67) on chromosome 8
(Fig. 6a). However, we could not find good candidate genes in the
candidate region (23.62–23.82 Mb) (Fig. 6b and Supplementary
Table 13). According to the pedigree information, the loss of awns
occurred at least twice in the varieties used here, suggesting that the
causal gene of the phenotype is multiallelic, as was the case for Hd1.
Therefore, we applied gene-based association analysis to this trait,
and found a strong peak close to but outside the 23.62–23.82 Mb
region (Fig. 6c,d and Supplementary Fig. 20). Among the genes
forming the peak, LOC_Os08g37890, which showed the sixth highest
−log10 P value (Fig. 6e and Supplementary Table 12), encodes a
member of epidermal patterning factor-like protein (EPFL) (Fig. 6f).
In Arabidopsis, one member of EPFL is involved in cell division in the
process of stomata formation50, although the roles of this gene family

Nature Genetics  VOLUME 48 | NUMBER 8 | AUGUST 2016 931

 Articles

a 8 b Hap. A
d Hd2
LOC_
Os11g08410
LOC_
5 Os01g62780 Hd6
6 Hd1
–log10 (P)

–log10 (P)
4 Hap. B and D
3

2 2

1
0
Hap. E and F
Chr. 6 (Mb) 7.0 7.5 8.0 8.5 9.0 9.5 0
1 2 3 4 5 6 7 8 9 10 11 12
Chr. 6 (Mb) 7.0 7.5 8.0 8.5 9.0 9.5 Chromosome
e
c LOC_Os06g16370 (Hd1) 5
Chr. 6
(+ strand) 4

–log10 (P)
3
+328 +744
r 2
Hap. A 36 bp del. –
2
Hap. B and D – – 1
Hap. E and F – 43 bp del.
0 0.2 0.4 0.6 0.8 1.0 0
Chr. 6 (Mb) 7.0 7.5 8.0 8.5 9.0 9.5
Figure 5  Analyses of the peak for days to heading on chromosome 6. (a) Local Manhattan plot (top) f
and LD heatmap (bottom) surrounding the peak on chromosome 6. Aarrow indicates the position of 5 Hd2: –log10 (P) = 4.84 (4th)
nucleotide variations in Hd1. (b) Schematic representation of the genome structure of the region in a.
© 2016 Nature America, Inc. All rights reserved.

LOC_Os11g08410: –log10 (P) = 4.67 (7th)

Hd6: –log10 (P) = 4.37 (12th)
Major and minor alleles on each polymorphic site are represented in blue and orange, respectively 4 LOC_Os01g62780: –log10 (P) = 3.85 (20th)
Hd1: –log10 (P) = 3.78 (22th)
(Online Methods). The varieties were divided based on Hd1 function as follows: haplotype (Hap.)

–log10 (P)
A (intermediate), Hap. B and D (functional), and Hap. E and F (null). Arrowhead indicates position of 3
the nucleotide polymorphisms in Hd1, which are not reflected in the graphical genotype owing to the
2
effect of surrounding nucleotide polymorphisms (Online Methods). (c) Exon-intron structure of Hd1 and
DNA polymorphisms in Hd1. (d) Manhattan plot of gene-based association analysis for days to heading. 1
Dashed line represents a 0.2 false discovery rate (−log 10 P = 3.02). (e) Local Manhattan plot of gene-
based association analysis surrounding the peak on chromosome 6. Arrow indicates the position of Hd1. 0
(f) Plot of −log10 P values of each marker. The markers were arranged in the descending order of −log 10 0 2 4 6 8 10 12 14
P values. Arrows indicate positions of genes identified in this study. Number of markers (thousands)

in rice are largely unknown. The varieties in this study had three
haplotypes, A, B and C (Fig. 6f). The alignment of LOC_Os08g37890
and homologs indicated that haplotype C might encode the entire
region of EPFL, whereas haplotype A (Nipponbare haplotype) pro-
duces a truncated protein owing to a 4-bp deletion in its coding
region. Furthermore, haplotype B contained a 6-bp deletion in the
region encoding two highly conserved amino acids (Supplementary
Fig. 21a–c). Consequently, haplotype C could be a functional allele,
npg
process that includes the selection of a not highly structured popu-
lation and whole-genome sequencing. The process includes: (i) the
detection of significant signals through association analysis with
individual nucleotide polymorphisms; (ii) the definition of candi-
date regions with significant signals based on LD; (iii) the extraction
of causal genes based on the function of polymorphisms, annota-
tion information including that of Arabidopsis homologs as the case
of LOC_Os01g62780 and (iv) transformation of a gain-of-function

whereas haplotype A and B were predicted to be loss-of-function
alleles. This prediction corresponded well to the phenotypic varia-
tion in these varieties (Fig. 6g); i.e., varieties carrying haplotype A
or haplotype B did not form awns whereas the varieties with
haplotype C developed awns. When we examined the genome struc-
ture of this region, the pattern in the LD block corresponded to the
predicted function of LOC_Os08g37890 (Fig. 6h). These observations
suggested that the strong peaks were spurious signals as in the case
of Hd1. To confirm that LOC_Os08g37890 is a causal gene for the
awnless phenotype, we introduced the entire genome of haplotype B
or haplotype C into Nipponbare (haplotype A). As expected, the
plants transformed with haplotype C (functional allele) formed long
awns, whereas plants carrying haplotype B or the vector control had
the awnless phenotype (Fig. 6i and Supplementary Fig. 21d–f).
These results indicated that LOC_Os08g37890 is a causal gene for
the awnless phenotype and demonstrated that gene-based association
analysis is an efficient method to deal with spurious signals caused
by allelic heterogeneity.
DISCUSSION
To assess the potential of GWAS for the rapid identification of new
genes associated with agronomic traits, we designed an experimental
haplotype into plants containing loss-of-function haplotype. As
expected, we identified scores of candidate genes in step ii. However,
at step 3, one to several candidates were identified in most of the cases.
As a consequence, although a large LD results in inclusion of many
polymorphisms in a candidate region, our results indicated that func-
tionally meaningful polymorphisms were less numerous and could
be evaluated by genetic transformation.
In the analysis of Hd1, we identified a spurious association caused
by allelic heterogeneity and complex genome structure (Fig. 5).
Considering the elaborate statistics, this is considered a serious prob-
lem for GWAS46,51,52. In this study, we demonstrated that gene-based
association analysis, which was facilitated by whole-genome sequencing,
is an effective approach for dealing with spurious associations
(Figs. 5 and 6). In crop domestication and breeding, it is not uncom-
mon to independently select alleles of the same gene, which results
in allelic heterogeneity of agronomically important genes41,53,54. This
suggests that gene-based association analysis is important for GWAS
of agronomic traits. In this context, there is a consideration impor-
tant to mention. When the number of functionally different alleles
in a gene is two, the functional difference can be explained by a SNP.
Under such a condition, the gene-based association approach results
in a decrease in statistical power. We propose the use of both single

932 VOLUME 48 | NUMBER 8 | AUGUST 2016  Nature Genetics

 Articles

a 25
c d e
LOC_Os08g37890:
15 15
20 –log10 (P) = 15.73 (6th)
–log10 (P)

15

–log10 (P)

–log10 (P)
–log10 (P)
15 10 10
10
10
5 5
5 5

0 0 0 0
1 2 3 4 5 6 7 8 910 11 12 1 2 3 4 5 6 7 8 9 10 11 12 0 2 4 6 8 10 12 14
Chr. 8 23.5 23.6 23.7 23.8 23.9 24.0
Chromosome Chromosome (Mb) Number of markers
(thousands)
b 25 f Chr. 8
LOC_Os08g37890
g 2013 2014
(+ strand)
20
5
* *
–log10 (P)

15 5
4

Awn length (cm)

Awn length (cm)
10 +236 +238 +297 4
5 Hap. A AGC (S) – – 3
3
0 Hap. B ACC (T) 6 bp del. 4 bp ins.
2 2
Chr. 8 23.5 23.6 23.7 23.8 23.9 24.0 Hap. C AGC (S) – 4 bp ins.
(Mb)
1 1
n.s. n.s.
h 0 0
Hap. A B C Hap. A B C
Hap. A (n = 140) (n = 21) (n = 15)
© 2016 Nature America, Inc. All rights reserved.

Hap. B
i VEC Hap. B Hap. C
r2
Hap. C
0 0.2 0.4 0.6 0.8 1.0 Chr. 8 (Mb) 23.5 23.6 23.7 23.8 23.9 24.0

Figure 6  GWAS for awn length and identification of the causal gene for the peak on chromosome 8.
(a) Manhattan plot of single-polymorphism-based association analysis. Dashed line represents a
significance threshold (−log10 P = 4.21). (b) Local Manhattan plot of single-polymorphism-based
association (top) and LD heatmap (bottom) surrounding the peak on chromosome 8. Arrow indicates
the position of nucleotide variations in LOC_Os08g37890. Dashed lines indicate the candidate region
for the peak. (c) Manhattan plot of gene-based association analysis. Dashed line represents a 0.2 false discovery rate (−log 10 P = 2.28). Arrowheads in a
and c indicate the position of strong peaks investigated in this study. (d) Local Manhattan plot of gene-based association analysis surrounding the peak on
chromosome 8. The arrow indicates the position of LOC_Os08g37890. (e) −log10 P values of each marker, arranged in descending order of −log10 P values.
Arrow indicates the position of LOC_Os08g37890. (f) Exon-intron structure of LOC_Os08g37890 and DNA polymorphisms in that gene. ins, insertion;
del, deletion. (g) Awn length based on the haplotypes for LOC_Os08g37890 in 2013 (left) and 2014 (right). Data are presented as shown in Figure 2d.
Differences between the haplotypes were statistically analyzed based on Tukey’s test (*P < 0.05; n.s., not significant). (h) Schematic representation of
the genome structure of the indicated region of chromosome 8. Major and minor alleles on each polymorphic site are represented in blue and orange,
respectively. (i) Awn lengths for transgenic plants transformed with empty vector (VEC), haplotype B (Hap. B) and haplotype C (Hap. C). Scale bar, 15 mm.
npg

polymorphisms and gene-based analysis combined with functional
annotation of each nucleotide polymorphism.
All the genes analyzed in the present study included polymorphisms
that induced amino acid exchanges. It should be reasonable to expect
that such polymorphisms have a higher probability of being function-
ally important. However, polymorphisms in regulatory regions often
contribute to phenotypic diversity in agronomic QTLs, such as qSH1,
GS5 and GL7 (refs. 55–57). To deal with such cases, the combined use
of GWAS with expression profiling data could be effective as recently
reported58. We are currently assessing that approach.
URLs. The rice cultivar database, http://ineweb.narcc.affrc.go.jp/;
Rice HapMap Project, http://www.ncgr.ac.cn/RiceHap2/; PHYLIP,
http://evolution.genetics.washington.edu/; PLINK, http://pngu.mgh.
harvard.edu/~purcell/plink/; ClustalW, http://clustalw.ddbj.nig.ac.jp/;
Ensembl Plants, http://plants.ensembl.org/Oryza_rufipogon/; DDBJ
Sequence Read Archive, http://trace.ddbj.nig.ac.jp/dra/.
Methods
Methods and any associated references are available in the online
version of the paper.
Accession codes. The sequence data has been deposited in
DNA Data Bank of Japan Sequence Read Archive (DRA):
DRA004358.
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
Acknowledgments
This work was supported by the Japan Society for the Promotion of Science
through a Grant in Aid for Scientific Research (A) (26252001), Council for Science,
Technology and Innovation (CSTI), Cross-Ministerial Strategic Innovation
Promotion Program (SIP), “Technologies for Creating Next-Generation
Agriculture, Forestry and Fisheries” (funding agency: Bio-oriented Technology
Research Advancement Institution, NARO), and by Grant in Aid for JSPS Fellows
Grant Number 16J08722.
AUTHOR CONTRIBUTIONS
K.Y., K.A., H.T., P.-C.L. and L.H. performed the field experiments and analyzed
the results. K.Y., K.A. and H.T. performed the genotyping and the genome data
analyses. M.Y. and S.Y. prepared the population material. K.Y. produced the
constructs and generated and analyzed the transformants. K.Y., E.Y., K.A., H.K.,
K.H. and M.M. designed the research and wrote the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.

Nature Genetics  VOLUME 48 | NUMBER 8 | AUGUST 2016 933

 Articles
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 ONLINE METHODS
Plant material and genotyping. We used a set of 176 japonica rice varieties
cultivated in Japan, which were collected from various places in Japan59,60,
and maintained in the Togo Field, Field Science Center, Nagoya University
(Supplementary Table 1). Total DNA was extracted from leaves of each variety
using the DNeasy Plant Mini Kit (Qiagen). The DNA was physically sheared
into ~500 bp fragments using Covaris S2 (Covaris). The fragmented DNA
was used for a DNA library construction with the NEBNext DNA Library
Prep Reagent Set for Illumina (BioLabs). The DNA library was sequenced
using an Illumina HiSeq 2000 (Illumina Co, Ltd.), and a total of 3.8 billion
paired-end 100-bp reads were obtained. All reads were mapped against
Os-Nipponbare-Reference-IRGSP-1.0 pseudomolecules using bwa-mem with
the −M option of BWA software61. The mapped reads were realigned using
RealignerTargetCreator and indelRealigner of GATK software62. To label SNPs
and indels, UnifiedGenotyper of GATK was used with the −glm BOTH option.
After removing nucleotide variations with missing rates ≥ 0.25 and minor
allele frequency < 0.05, we identified 426,337 SNPs and 67,544 indels. All
nucleotide polymorphisms were categorized based on their location in the ref-
erence genome (groups I−V). We used the generic feature format file version 3
(gff3) from the MSU rice genome annotation project63 for information on gene
position and coding sequence. With respect to categorizing polymorphisms,
we regarded the promoter region as the 2 kb area upstream of the translation
initiation site of each gene.
© 2016 Nature America, Inc. All rights reserved.
Population genetic analyses. The population structure of the 176 varieties was
estimated using PCA performed by using the software EIGENSTRAT64. To
analyze the genetic relationship between the 176 varieties and other japonica
rice varieties, we used the genotype data set obtained from temperate and trop-
ical japonica varieties as described20 (Rice HapMap Project; see URLs). Genetic
relationships were estimated using neighbor-joining trees constructed using
the software PHYLIP version 3.695 (see URLs). The LD between SNPs and
indels in the 176 varieties was evaluated using squared Pearson’s correlation
coefficient (r2) as calculated with the −r2 command in the software PLINK ver-
sion 1.9 (see URLs). The LD heatmaps surrounding peaks in the GWAS were
constructed using the R package “LDheatmap”65. We estimated the candidate
regions using an r2 > 0.6 (refs. 66–68).
Phenotyping. Phenotyping of agronomic traits was performed in 2013 and
2014 at the paddy field located at the Togo Field, Field Science Center, Nagoya
University. Heading date was recorded as the appearance date of the first pani-
cle and days to heading as the number of days from sowing to heading dates.
Plant height is the mature length of main culm from the soil surface to the
tip of the panicle. Panicle length of the main culm was used to represent the
npg
panicle length of a plant. Panicle number per plant was measured 20 days
after the initiation of flowering. Leaf blade width was measured at the wid-
est point of flag leave sampled from the main stem. The awn length of apical
spikelets on each primary branch was used to represent the awn length of the
whole panicle. Data for each trait were evaluated from three randomly chosen
plants per variety.
GWAS. For GWAS, we used a linear mixed model (LMM). The LMM assumes
the following model69:
y = X b + Zu + e (1)
where y is a vector of phenotype; X is a matrix of fixed effects including the
nucleotide polymorphism (for single-polymorphism-based association analy-
sis) or gene haplotype (for gene-based association analysis, see below), the
grand mean and principal component 1 calculated in the genetic relationship
analysis. β is a vector of effects; Z is an incidence matrix relating y to u. The
variable u models the genetic background of each line as a random effect with
u ~ N(0, Kσ2G), where K is a kinship matrix calculated from the nucleotide
polymorphisms, and σ2G is the genetic variance. ε is a matrix of residual effects
such that ε ~ N(0, Iσ2e), where I is an identity matrix and σ2e is the residual
variance. For gene-based association analysis, we performed gene haplotype
identification based on polymorphisms localized on the coding regions by an
R script written in house. Then the difference in haplotype was represented by
doi:10.1038/ng.3596
dummy variables. We modified the R package “rrBLUP” version 4.3 (ref. 70) to
enable the use of a matrix of dummy variable as fixed effects included in X of
equation (1). The genome-wide significance threshold was determined using
permutation-based false-discovery-rate-adjusted P values71. The permutation
tests were repeated 1,000 times.
Graphical genotype visualization. To display genome structure in the tested
populations schematically, we generated the graphical genotype. Because the
genotype data set was too large scale for presentation using the graphical
genotype, we used the following method to compress the data. In regions of
interest, polymorphisms were converted to numeric genotypes represented by
1 and −1, which corresponded to major and minor polymorphisms, respec-
tively. Polymorphisms were then divided into bins (bin size = 10 kb) based on
their location in the genome. In each bin, the tested population was forcibly
clustered into two groups using the k-means clustering algorithm conducted
using the R function “kmeans”, and the information on the cluster was used
for generating the graphical genotype. In each bin, major and minor clusters
were represented in blue and orange in the graphical genotype.
Phylogenetic analysis. Amino acid alignment of sequences was conducted
using ClustalW (see URLs) from the DNA Data Bank of Japan (DDBJ) with
default parameter settings and then manually adjusted to optimize alignments.
Phylogenetic trees were constructed using the neighbor-joining algorithm in
MEGA version 6 (ref. 72) with 1,000 replicates using the following parameters:
gaps/missing data, complete deletion; Jones-Taylor-Thornton (JTT) model;
pattern among lineages, same; and rates among sites, uniform.
Transgenic analysis. Full-length genomic DNAs encompassing the entire
sequence of LOC_Os01g62780 were amplified from Nipponbare (Hap. A) and
Line92 (Hap. B) using PCR. Similarly, LOC_Os11g08410 full-length genomic
DNAs were amplified from Nipponbare (Hap. A) and Line9 (Hap. C). For LOC_
Os08g37890, full-length genomic DNAs were amplified from Line7 (Hap. B)
and Line154 (Hap. C). These PCR products were cloned into pENTER/D-
TOPO (Invitrogen). DNA fragments were then subcloned into the Gateway
binary vector (pGWB) using Gateway LR Clonase Enzyme mix (Invitrogen).
For constitutive expression of LOC_Os04g52479, the coding sequences of
LOC_Os04g52479 were amplified from cDNA of Nipponbare (Hap. A) and
Line130 (Hap. B), and PCR products were cloned into pENTER/D-TOPO.
The LOC_Os04g52479 cDNA fragments were subcloned using the LR reac-
tion into the Gateway binary vector pUBQ-pGWB, which contains the maize
ubiquitin promoter upstream of the cloning site. The primer sets used for PCR
are listed in Supplementary Table 14. The DNA constructs and empty vectors
that served as controls were introduced into Nipponbare using Agrobacterium
tumefaciens (EHA105)-mediated transformation, according to ref. 73. More
than 12 independent T0 plants were generated and grown to maturity in pots
under greenhouse conditions.
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