Supplementary information
A natural variant of NAL1, selected in high-yield rice breeding programs, pleiotropically
increases photosynthesis rate
Toshiyuki Takai1,3*, Shunsuke Adachi2,3*, Fumio Taguchi-Shiobara3, Yumiko Sanoh-Arai1, Norio
Iwasawa1, Satoshi Yoshinaga1, Sakiko Hirose1, Yojiro Taniguchi1, Utako Yamanouchi3, Jianzhong
Wu3, Takashi Matsumoto3, Kazuhiko Sugimoto3, Katsuhiko Kondo3, Takashi Ikka3, Tsuyu Ando4,
Izumi Kono4, Sachie Ito4, Ayahiko Shomura4, Taiichiro Ookawa2, Tadashi Hirasawa2, Masahiro
Yano3, Motohiko Kondo1 & Toshio Yamamoto3
1
NARO Institute of Crop Science, Tsukuba, Ibaraki 305-8508, Japan.
2
Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo
183-8509, Japan.
3
National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
4
Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, Tsukuba,
Ibaraki 305-0854, Japan.
*Toshiyuki Takai and Shunsuke Adachi contributed equally to this work.
Corresponding author: T. Yamamoto, National Institute of Agrobiological Sciences, Tsukuba,
Ibaraki 305-8602, Japan. Tel.: +81-29-838-7135; Fax: +81-29-838-7468. E-mail:
toshyama@affrc.go.jp
a
b
Number of lines
30
30
Koshihikari Takanari
Koshihikari Takanari
20
20
10
10
0
16
0
22
28
34
40
c
0
1
2
3
4
5
6 7
8
9 10 11 12
22
16
28
34
40
Photosynthesis rate
(μmol m-2 s-1)
Photosynthesis rate
(μmol m-2 s-1)
d
0
20
1 2
3
4
5 6
7
8 9 10 11 12
20
GPS
40
40
60
60
80
80
100
(cM)
100
(cM)
Supplementary Figure S1. QTL analysis of photosynthesis rate. (a, b) Frequency distributions of
photosynthesis rate of flag leaves at full heading stage for 82 BILs in the Koshihikari background (a) and 86
BILs in the Takanari background (b). Yellow-green arrowheads indicate mean photosynthesis rates for
Koshihikari; dark green, Takanari. (c, d) QTL mapping of photosynthesis rate in Koshihikari-background BILs
(c) and Takanari-background BILs (d). Triangles and boxes to the right of each chromosome represent LOD
peaks of putative QTLs and their 1-LOD support intervals, respectively. Upward triangles indicate that leaf
photosynthesis was increased by the Takanari allele; downward triangles, increased by the Koshihikari allele.
60
50
Photosynthesis rate (μmol m -2 s-1 )
40
30
20
10
0
Koshihikari
Koshihikari NIL-GPS
Takanari
Takanari NIL-GPS
-10
60
50
40
30
20
10
0
-10
0
200
400
600
800
1,000
1,200
1,400
0
200
400
600
800
1,000
1,200
1,400
-1
Intercellular CO2 concentration (μmol mol )
Supplementary Figure S2. Fitting of the Farquhar model29 to the photosynthesis–intercellular CO2
concentration response from Fig. 3. Solid lines indicate photosynthesis rate limited by RuBP carboxylation by
Rubisco; dotted lines indicate photosynthesis rate limited by RuBP regeneration. Red circles indicate
photosynthesis rate at an ambient CO2 concentration of 390 μmol mol−1.
Vc,max (μmol m -2 s-1)
270
R =0.99**
240
210
Koshihikari
Koshihikari NIL-GPS
Takanari
Takanari NIL-GPS
180
150
2.0
3.0
4.0
-2
5.0
Rubisco content (g m )
Supplementary Figure S3. Relationship between Rubisco content and maximum rate of carboxylation (Vc,max)
among Koshihikari, Takanari, and the reciprocal NIL-GPS. Vc,max was estimated from the model-fitting analysis
in Supplementary Fig. S2. **P < 0.01.
a
Takanari
NIL - GPS
0
a
Takanari
250
a
Koshihikari
NIL - GPS
500
a
Koshihikari
a
15
Stomatal length (μm)
a
b
10
5
0
Takanari
NIL - GPS
Takanari
NIL - GPS
Takanari
Koshihikari
NIL - GPS
0.5
750
Takanari
1.0
b
Koshihikari
NIL - GPS
a
b
a
c
1,000
Koshihikari
-2
b
1.5
0.0
Stomatal density (no. mm )
b
2.0
Koshihikari
Stomatal conductance
-2 -1
(mol m s )
a
Supplementary Figure S4. Effects of GPS alleles on stomatal characteristics. Comparisons of stomatal
conductance (a), density (b), and length (c) in flag leaves at full heading stage in Koshihikari, Koshihikari
NIL-GPS, Takanari, and Takanari NIL-GPS. Each column represents mean ± s.d. (n = 9). Different letters
indicate significant difference at the 5% level (Tukey–Kramer HSD test).
i
d
Leaf width (cm)
2.0
c
a
b
1.0
***
50
25
0
a
b
20
10
Takanari
NIL - GPS
Takanari
Koshihikari
NIL - GPS
Koshihikari
Takanari
NIL - GPS
0
Takanari
Koshihikari
NIL - GPS
Koshihikari
0.5
0.0
100
c
a
30
0
k
40
100
a
RNAi- NAL1
Koshihikari
j
2.5
1.5
10
0
RNAi- NAL1
Koshihikari
0.5
20
***
200
a
b
b
Takanari
NIL - GPS
***
***
75
300
Takanari
1.0
Culm length (cm)
1.5
30
T65 T65-nal1
h
100
RNAi- NAL1
Leaf length (cm)
Leaf width (cm)
2.0
g
40
0
T65 T65-nal1
75
50
25
0
Koshihikari
f
2.5
0.0
0
Leaf length (cm)
e
25
T65 T65-nal1
T65 T65-nal1
***
100
Koshihikari
0
0.0
***
50
200
Koshihikari
NIL - GPS
10
SLA (cm2 g-1)
***
SLA (cm2 g-1)
***
0.5
20
Culm length (cm)
1.0
75
300
RNAi- NAL1
1.5
30
d
100
Culm length (cm)
2.0
c
40
Koshihikari
b
2.5
Leaf length (cm)
Leaf width (cm)
a
Supplementary Figure S5. Comparisons of morphological traits between lines differing in NAL1 (GPS)
genotype or expression. (a–d) Comparisons of flag leaf width (a), flag leaf length (b), culm length (c), and
specific leaf area (SLA) (d) in Taichung 65 and nal1 mutant line (T65-nal1). Each column represents mean ±
s.d. (n = 10); ***P < 0.001 versus T65 (Student's t-test). (e–h) Comparisons of flag leaf width (e), flag leaf
length (f), culm length (g), and SLA (h) in Koshihikari and transgenic plants with RNAi-induced suppression of
NAL1 (RNAi-NAL1). Each column represents mean ± s.d. (n = 25 for RNAi-NAL1, n = 5 for Koshihikari);
***P < 0.001 versus Koshihikari (Student's t-test). (i–k) Comparisons of flag leaf width (i), flag leaf length (j)
and culm length (k) in Koshihikari, Koshihikari NIL-GPS, Takanari, and Takanari NIL-GPS. Each column
represents mean ± s.d. (n = 9). Different letters indicate significant difference at the 5% level (Tukey–Kramer
HSD test).
108, normal
70ins
97
144del
95
70ins
25ins
53
25ins
43
70,15ins
97
70ins
136del
25ins
25
52ins
18
8ins
144del
17
70ins
25ins
29del
14
25ins
175del
13
337ins
144del
11
52ins
25ins
10
135del
10
175del
7
2k ins
93ins
5
25ins
144del
3
5.8k ins
93ins
3
2k ins
93ins
25ins
2
70,15ins
25ins
2
29del
2
70ins
25ins
29del
1
175del
8ins
1
Total 572
Supplementary Figure S6. Schematic diagram of spliced transcripts for NAL1 in Koshihikari. Orange bars and
triangles represent inserted segment. White bars in exons represent deleted segment. Orange triangles indicate
that inserted segment was derived from retrotransposon. A total of 572 transcripts was sequenced and the
number of each variant is shown at the right side.
408, normal
25ins
35
718ins
29
116del
20
29del
18
93ins
718ins
155ins
337ins
16
99del
13
15ins
10
337ins
7
8ins
7
718ins
175del
5
718ins
337ins
5
27ins
4
99del
99del
2
25ins
99del
2
93ins
2
93ins
718ins
337ins
2
175del
2
155ins
337ins
2
251del
3ins
241del
144del
2
99del
136del
1
93ins
718ins
25ins
337ins
1
11ins
1
29del
718ins
1
25ins
29del
1
337ins
11del
337ins
25ins
1
1
27ins
25ins
1
25ins
265del
1
52del
144del
25ins
21del
1
7del
1
11del
1
15ins
29del
1
Total 604
Supplementary Figure S7. Schematic diagram of spliced transcripts for NAL1 in Takanari. Orange bars and
triangles represent inserted segment. White bars in exons represent deleted segment. Orange triangles indicate
that inserted segment was derived from retrotransposon. A total of 604 transcripts was sequenced and the
number of each variant is shown at the right side.
Supplementary Table S1. Putative QTLs controlling leaf photosynthesis of flag leaves
at full heading stage in reciprocal BILs between Koshihikari and Takanari.
Genetic background
Koshihikari
Takanari
a
b
Chr.
Flanking marker
LOD
Aa
R2b
3
4
1
3
4
5
RM1371
RM3534
RM7594
RM3204
RM3534
RM3838–RM1386
4.0
3.6
4.0
3.8
4.0
3.5
–1.4
2
–1.7
3.0
1.7
1.5
13.4
12.1
13.5
16.9
13.9
13.6
Additive effect of the Takanari allele compared with the Koshihikari allele.
Percentage of phenotypic variance explained by each QTL.
Supplementary Table S2. Sequence polymorphism in the genomic region of NAL1
among Takanari, 14 cultivars in the pedigree of Takanari, and Koshihikari.
Intron 2
Exon 1
Exon 2
5895-bp
A/C
C/G
T/G
G/A
C/T
retrotransposon
Variety
S.S.
690
860
1328
1405
1466
164a
Takanari
A
C
T
G
C
Milyang42
A
C
T
G
C
Milyang25
A
C
T
G
C
Tongil
A
C
T
G
C
IR24
A
C
T
G
C
IR8
A
C
T
G
C
IR4-93-2
A
C
T
G
C
Mudgo
A
C
T
G
C
IR262-43-8-11
A
C
T
G
C
Peta
A
C
T
G
C
H4
A
G
T
G
C
Taichung Native 1
C
G
G
G
T
Jinheung
C
G
G
A
T
Dee-geo-woo-gen
C
G
G
A
T
Yukara
C
inserted
G
G
A
T
Koshihikari
C
inserted
G
G
A
T
S.S., synonymous substitution.
a
b
Position from start of the 5' UTR based on Takanari genome.
32
The 30-bp deletion is present in the nal1 mutant .
Exon 3
G/A
Arg/His
1640
G
G
G
G
G
G
G
G
G
G
G
G
G
G
A
A
Exon 4
30-bp
deletionb
2402
-
Exon 5
3' UTR
C/T
G/A
A/G
C/T
Ala/Val
2858
C
C
C
C
C
C
C
C
C
C
C
C
T
T
T
T
Val/Ile
2884
G
G
G
G
G
G
G
G
G
G
G
G
A
A
A
A
3318
A
A
A
A
A
A
A
A
A
A
A
A
A
A
G
G
3362
C
C
C
C
C
C
C
C
C
C
C
T
T
T
T
T
Supplementary Table S3. Primer sequences used in this study.
Name
Upper primer (5'–3')
Lower primer (5'–3')
Primers used for mapping of the GPS locus
InDel_4_103
CCTCAAGGACCGTAAAATTGCTC
GGCAATTAATTAGCATCTCTTCTCTCC
InDel_4_104
TGCCGTCTTTCTTGAGTTCTTGC
AACAAACAACTCAGCCGCCATC
InDel_4_105
TGCAGGTTTCCTTCAGCTTTGTC
ACCAAAATATCGCCCAAAATTGC
InDel_4_106
CCCGTTGTCACTATCCCAACTCA
TCCCATAAAGCAAACCTCACACG
InDel_4_135
ACAACTTTCGGGTGGACCAATCT
TGTGCTATGCCATTTGCCAATCT
Primers used for making the RNAi construct
NAL1_RNAi
CTGGAACTGAAGGGGAGGAG
ACTTTTGGCTGTCCACAATGAC
Primers used for quantitative RT-PCR
NAL1-F2R2
TGGGTGCACACACACAGAGA
TGCACAATGCTGTAGGTTGGA
UBQ2
GAGCCTCTGTTCGTCAAGTA
ACTCGATGGTCCATTAAACC
Primers used for transcript analysis
NAL1-F1R7
CGCTTTCGGCATTCGTTATC
GGGATCCGGCAATGGTGTAT
Primers used for detecting sequence polymorphism in the genomic region of NAL1
GPS-01
CGCTTTCGGCATTCGTTATC
AGGTTGGATGTCGGCCATAA
GPS-02
CATAGCAAGATATTGCGGCGTTT
TGCTTAATGCACATGGTATTTTTGC
GPS-03
GGCTCAAACACCTAAAGAGCAAA
AACTCCTTCCAATCTCCGATCA
GPS-04
TCTTGTGCAGATTAAAGCTTCTGG
GGGTTTGTTCCAGCATAGATTCC
GPS-05
TGCAAAAAGTGCTGGTTCTGAATTT
TCCGGCAATGGTGTATATCAGGT
GPS-06
CATGGCCCTGAAAACTGGAC
GATGCCTCCTCCCCTTCAGT
GPS-07
CGCCATGTCGCTCCATCT
TCATGAGCACAGCAAAACTGC