control of root system architecture by deeper rooting 1 ......kp‒yfp (expected size 59.0 kda) and...

1

Supplementary Information File

Control of root system architecture by DEEPER ROOTING 1 increases rice

yield under drought conditions

Yusaku Uga*, Kazuhiko Sugimoto, Satoshi Ogawa, Jagadish Rane, Manabu Ishitani,

Naho Hara, Yuka Kitomi, Yoshiaki Inukai, Kazuko Ono, Noriko Kanno, Haruhiko

Inoue, Hinako Takehisa, Ritsuko Motoyama, Yoshiaki Nagamura, Jianzhong Wu,

Takashi Matsumoto, Toshiyuki Takai, Kazutoshi Okuno & Masahiro Yano

*To whom correspondence should be addressed. E-mail: [email protected]

This PDF file includes:

References, Supplementary Figures 1–26, and Supplementary Tables 1–4

Nature Genetics: doi:10.1038/ng.2725

2

References

52. Uga, Y., Fukuta, Y., Ohsawa, R. & Fujimura, T. Variations of floral traits in

Asian cultivated rice (Oryza sativa L.) and its wild relatives (O. rufipogon Griff.).

Breed. Sci. 53, 345–352 (2003).

53. van Genuchten, M.T. A closed-form equation for predicting the hydraulic

conductivity of unsaturated soils. Soil Sci. Soc. Am. J. 44, 892–898 (1980).

54. Lilley, J.M. & Ludlow, M.M. Expression of osmotic adjustment and dehydration

tolerance in diverse rice lines. Field Crops Res. 48, 185–197 (1996).


Supplementary Figure 1. Representative vertical root distribution of IR64, Dro1-NIL, and Kinandang Patong in an upland field. Root distribution was assessed by using the trench method. Yellow dashed lines indicate the extent of root elongation.

IR64 Dro1-NIL Kinandang Patong

20 cm

40 cm

60 cm

0 cm


Supplementary Figure 2. Estimation of vertical root distribution of Dro1-NIL, IR64, and Kinandang Patong by the monolith sampling method. (a–c) Steps in the monolith sampling method. (a) Driving the monolith sampler into the ground. (b) Pulling the monolith sampler from the ground. (c) Dividing the soil monolith into 12 parts. (d) Typical root distribution of each line obtained by using the monolith sampler. The photos in d were taken to show the condition of root samples obtained by using the sampler. In practice, each soil monolith was divided into 12 blocks (A to L), and root dry weight in each block was measured.

A B

C

D

E F

G

H

I J

K

L

5 cm

5 cm

10 cm

10 cm

10 cm

IR64 Kinandang Patong Dro1-NIL 10 cm 10 cm

d

a b c


Supplementary Figure 3. Effect of DRO1 on root dry weight in different soil layers and on shoot traits in an upland field. (a) Horizontal and vertical root distribution throughout the soil profile as indicated by root dry weight. Letters A–L indicate the position of roots in the soil monolith block as illustrated in Supplementary Figure 2d. (b) Total root dry weight of A–L blocks per plant, shoot length, tiller number per plant, and shoot dry weight per plant. Data are means ± s.d.; n = 10 plants (Dro1-NIL and KP); n = 9 plants (IR64). Values labeled with different letters differ significantly among the three lines (P <0.05, Tukey’s HSD test). KP, Kinandang Patong.

b

Tota

l roo

t dry

wei

ght (

g)

a a

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

IR64 Dro1-NIL

KP

0

20

40

60

80

100

120

140

IR64 Dro1-NIL

KP

Shoo

t len

gth

(cm

)

a a

b

0

50

100

150

200

250

IR64 Dro1-NIL

KP

Tille

r num

ber p

er p

lant

a a

b

0

50

100

150

200

250

IR64 Dro1-NIL

KP

Shoo

t dry

wei

ght p

er p

lant

(g)

a a

b

b

0.0 0.5 1.0 1.5 2.0 2.5

H

G

F

E

Root dry weight (g)

c

a b

b

a a

a a

b

a a

0.00 0.05 0.10 0.15 0.20 0.25 0.30

L

K

J

I

Root dry weight (g)

a

a b

c

a b

c

a b

b

a a

a

b

0.00 0.05 0.10 0.15 0.20 0.25 0.30

D

C

B

A

IR64 Dro1-NIL KP

Root dry weight (g)

ab

a b

c

a b

b

a a

b

a b

Roo

t pos

ition

in

soil

mon

olith

blo

ck


Supplementary Figure 4. Difference in root growth angle between IR64 and Dro1-NIL grown in a greenhouse. (a) The root growth angle (θrga) of each plant was determined by measuring the angle between the soil surface (horizontal line) and the shallowest nodal root. (b) Mean root growth angle of IR64 and Dro1-NIL at around 30 days after sowing. Data are means ± s.d.; n = 20 plants. P values are based on Student’s t-test.

θrga

a b

0

10

20

30

40

50

60

70

IR64 Dro1-NIL

Roo

t gro

wth

ang

le (θ

rga)

P < 0.0001


Supplementary Figure 5. Effect of DRO1 on root and shoot morphological traits in a hydroponic system. Data are means ± s.d.; n = 20 plants. Number above each error bar indicates P value calculated by Student’s t-test. Maximum root length, crown root number, and root dry weight of IR64 and Dro1-NIL plants grown in hydroponic culture were measured as described previously9.

0

5

10

15

20

25

30

35

7 14 21 28 35 42

IR64 Dro1-NIL

Max

imum

root

leng

th (c

m)

Days after sowing

0.0255

0.0011 0.0170

0.0003 0.0422

0.4070

0

10

20

30

40

50

60

70

7 14 21 28 35 42

Shoo

t len

gth

(cm

)

Days after sowing

0.8858

0.8431

0.6134 0.1130

0.6364

0.9913

0

10

20

30

40

50

60

70

14 21 28 35 42

Cro

wn

root

num

ber

Days after sowing

0.1885

0.9553

0.5073

0.7610 0.0019

0.00

0.05

0.10

0.15

0.20

0.25

0.30

7 14 21 28 35 42

Roo

t dry

wei

ght (

g)

Days after sowing

0.2432 0.4586

0.3864

0.0664

0.1549

0.3231

0

1

2

3

4

5

6

7

8

21 28 35 42

Tille

r num

ber

Days after sowing

1.0000

0.1627

0.1172

0.0090

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

7 14 21 28 35 42

Shoo

t dry

wei

ght (

g)

Days after sowing

0.5974 0.5607

0.3547

0.1124

0.2467

0.0361


RM24393 RM7424

9S 9L

Recombinants

Ratio of deep rooting (%)

#6309-2-14-42

#6309-2-16-32

#6309-17-14-47

IR64-homo

KP-homo

11.9 + 4.8

62.0 + 6.1

6.4 + 3.5

12.4 + 4.5

63.7 + 7.6

INDEL09 CAPS05

candidate region for DRO1

6.0 kbp

Os09g0439800

8.7 kbp-fragment for complementation test

NotI KpnI

Supplementary Figure 6. Positional cloning of DRO1. (a) Candidate region of DRO1 locus reported previously10. CEN, centromere. (b) Physical map of region around DRO1 gene on chromosome 9. (c) Graphical genotypes and phenotypes of BC3F2 plants containing recombination between marker loci CAPS05 and INDEL09. Blue, homozygous for IR64 allele; red, homozygous for Kinandang Patong (KP) allele; yellow, heterozygous. IR64-homo and KP-homo are BC3F2 plants homozygous for alleles from IR64 and KP, respectively, in the entire candidate region. Mean ± s.d. of ratio of deep rooting is shown to the right of each graphical genotype. The DRO1 genotype was estimated for each recombinant and control line based on the results of Dunnett’s test at the 0.1% significance level (the phenotype of IR64-homo was used as the reference in this test).

CEN

IR64

KP

IR64

IR64

KP

Genotype of DRO1

a

b

c

DRO1

Controls


Supplementary Figure 7. Comparison of deduced amino acid sequences between DRO1 and its homologs in other plants. DRO1-kp, DRO1 of rice cv. Kinandang Patong. DRO1-ir, DRO1 of rice cv. IR64. DRO1-np, DRO1 of rice cv. Nipponbare. Non-rice sequences are XM_003578131.1 (Brachypodium distachyon), scaffold543596 18.8 (Hordeum vulgare), XM_002462405 (Sorghum bicolor), EU969762 (Zea mays), XM_002516205.1 (Ricinus communis), LOC100808146 (Glycine max), LOC100249804 (Vitis vinifera), LOC101207004 (Cucumis sativus), At1g72490 (Arabidopsis thaliana), XM_002326096.1 (Populus trichocarpa), and MTR_8g021200 (Medicago truncatula). Amino acid residues of DRO1 homologs identical to those in DRO1-kp (gray shading) are indicated by black shading. Amino acid residues identical across all accessions are boxed in yellow rectangles. The red arrowhead at the bottom of the figure indicates the position of the 1-bp deletion in IR64 relative to Kinandang Patong. Black bars above the sequences indicate putative N-myristoylation sites predicted by Motif Scan (http://myhits.isb-sib.ch/cgi-bin/motif_scan).

DRO1-kp

XM_003578131.1 scaffold543596 18.8 XM_002462405 EU969762

DRO1-np DRO1-ir

XM_002516205.1 LOC100808146 LOC100249804 LOC101207004 At1g72490 XM_002326096.1 MTR_8g021200

DRO1-kp

XM_003578131.1 scaffold543596 18.8 XM_002462405 EU969762

DRO1-np DRO1-ir


DRO1-kp

XM_003578131.1 scaffold543596 18.8 XM_002462405 EU969762

DRO1-np DRO1-ir


DRO1-kp

XM_003578131.1 scaffold543596 18.8 XM_002462405 EU969762

DRO1-np DRO1-ir


Mon

ocot

D

icot

1-bp deletion in IR64


Supplementary Figure 8. Subcellular localization of DRO1 in rice protoplasts transformed with CaMV35S::DRO1-kp::YFP or CaMV35S::DRO1-ir::YFP via electroporation. (a) In rice protoplasts transformed with CaMV35S::DRO1-kp::YFP, YFP fluorescence was visible in the cell membrane under a confocal laser scanning microscope. In protoplasts transformed instead with CaMV35S::DRO1-ir::YFP or CaMV35S::YFP (control), YFP fluorescence was visible in the cell nucleus and cytoplasm as well. Left, YFP fluorescence; center, merged YFP and (differential image contrast) DIC images; right, DIC alone. Scale bar, 10 μm. (b) Structure of the PUbi::myc::DRO1-kp::YFP and PUbi::myc::DRO1-ir::YFP constructs used for western analysis. (c) myc‒DRO1-kp‒YFP (expected size 59.0 kDa) and myc‒DRO1-ir‒YFP (expected size 56.6 kDa) fusion proteins produced in rice protoplasts were detected by western analysis using an anti-myc antibody. As shown in b, both constructs encode a fusion protein with a C-terminal YFP addition. Although a protein was produced by the PUbi::myc::DRO1-ir::YFP construct in this assay (c), it is possible that the DRO1-ir transcript in whole plants is targeted for nonsense-mediated decay because of the premature stop codon.

DRO1-ir::YFP

DRO1-kp::YFP

YFP(control)

DIC Merge YFP

a

c

CBB

Anti-myc

75 kDa

DRO1-kp::YFP

DRO1-ir::YFP

b

Ubq promoter ADH- 3×myc DEST Ter Dro1-kp YFP ter

Ubq promoter ADH- 3×myc Ter Dro1-ir YFP ter


28 (V)

27 (KPg-Sc)

11 (V, T3, n = 43) 28 (V, T3, n = 44)

0 10 20 30 40 50 60 70 80 90 100

0

10

20

30

0

10

20

30

Num

ber o

f pla

nts

Root growth angle (θrga)

21 (KPg-Sc, T3, n = 44)

27 (KPg-Sc, T3, n = 46) R

oot a

ngle

of c

urva

ture

(θra

c)

Inclination angle of root (θiar)

y(11) = 6.1078e0.0121x

r²(11) = 0.5304

y(28) = 14.992e0.0063x

r²(28) = 0.1028

y(21) = 9.9803e0.0142x

r²(21) = 0.8333

y(27) = 8.1951e0.0149x

r²(27) = 0.3421

0

10

20

30

40

50

60

70

50 60 70 80 90 100 110 120 130 140 150

a

b

c

Supplementary Figure 9. Root growth angle and gravitropic response in transgenic plants with a single copy of DRO1-kp and in vector control plants. (a) Root growth of transgenic plants at 2 days after sowing in agarose gel. KPg-Sc, plants containing a single copy of the 8.7-kb DRO1 genomic fragment from KP; (V) control plants transformed with an empty vector. (b) Distribution of root growth angle (measured from horizontal) in lines of KPg-Sc and V. V 11, mean ± s.d. = 49.9º ± 16.3º; V 28, mean ± s.d. = 56.2º ± 15.9º; KPg-Sc 21, mean ± s.d. = 78.3º ± 5.3º; KPg-Sc 27, mean ± s.d. = 80.0º ± 5.9º. (c) Relationship between inclination angle of root (θiar) and root angle of curvature (θrac) after rotation in transgenic DRO1-kp and vector control plants. θiar, inclination angle of root, i.e., the angle between the root and the horizontal immediately after rotation; θrac, root angle of curvature at 4 h after rotation.

11 (V, T3, n = 41) 28 (V, T3, n = 42) 21 (KPg-Sc, T3, n = 44) 27 (KPg-Sc, T3, n = 41)

( ) ( ) ( ) ( )


a

b

c

1 (RNAi)

3 (KPg-Mc)

Root growth angle (θrga)

Num

ber o

f pla

nts

0

10

20

30

40

0

10

20

30

40

1 (RNAi, T1, n = 39) 2 (RNAi, T1, n = 44)

0 10 20 30 40 50 60 70 80 90 100

3 (KPg-Mc, T3, n = 40) 9 (KPg-Mc, T3, n = 34)

Roo

t ang

le o

f cur

vatu

re (θ

rac)

Inclination angle to root (θiar)

Supplementary Figure 10. Root growth angle and gravitropic response in transgenic plants with multiple copies of DRO1-kp or an RNAi knockdown cassette. (a) Root growth of transgenic plants at 2 days after sowing in agarose gel. RNAi, plants containing an RNAi knockdown cassette; KPg-Mc, transformed plants containing multiple copies of the DRO1 genomic fragment from Kinandang Patong. (b) Distribution of root growth angle (measured from horizontal) in lines of RNAi and KPg-Mc. RNAi 1, mean ± s.d. = 59.3º ± 14.5º; RNAi 2, mean ± s.d. = 61.2º ± 11.7º; KPg-Mc 3, mean ± s.d. = 83.0º ± 3.5º; KPg-Mc 9, mean ± s.d. = 78.1º ± 7.0º. (c) Relationship between inclination angle of root (θiar) and root angle of curvature (θrac) after rotation in lines of RNAi and KPg-Mc. θiar, inclination angle of root, i.e., the angle between the root and the horizontal immediately after rotation; θrac, root angle of curvature at 4 h after rotation.

y(1) = 13.367e0.0092x

r²(1) = 0.0938

y(2) = 6.6123e0.0155x

r²(2) = 0.6745

y(3) = 11.337e0.0169x

r²(3) = 0.7431

y(9) = 12.63e0.013x

r²(9) = 0.6278

0 10 20 30 40 50 60 70 80 90

100

50 60 70 80 90 100 110 120 130 140 150

1 (RNAi, T1, n = 38) ( ) 2 (RNAi, T1, n = 44) ( ) 3 (KPg-Mc, T3, n = 42) ( ) 9 (KPg-Mc, T3, n = 34) ( )


Supplementary Figure 11. Expression patterns of DRO1 in tissues of Dro1-NIL. (a) In situ hybridization with a DRO1-specific sense probe on a longitudinal section of the root tip of Dro1-NIL at 30 days after sowing. (b–d) In situ hybridization of DRO1 near the basal part of the shoot at 4 days after sowing. (b, d) Hybridization with an antisense probe; (c) hybridization with a sense probe. (b, c) Longitudinal section; (d) transverse section.

b c a

d


0

1

2

3

4

5

6

7

8

9

10

SPL SPW STL SYL ANL

Supplementary Figure 12. Sizes of floral organs in IR64 and Dro1-NIL. SPL, spikelet length; SPW, spikelet width; STL, stigma length; SYL, style length; ANL, anther length. Data are means ± s.d.; n = 50. P values are based on Student’s t-test. We grew IR64 and Dro1-NIL in a paddy field of NIAS in the summer of 2012. Before flowering, a total of 50 spikelets were randomly collected from 10 plants in each line. The length and width of each spikelet and the lengths of the stigma, style, and anther were measured as described previously52.

P = 0.9127

P = 0.1527

P = 0.0057 P = 0.0221

P < 0.0001

IR64 Dro1-NIL

(mm)


Supplementary Figure 13. Effects of auxin (2,4-D) and cycloheximide (CHX) on DRO1 transcript levels in Dro1-NIL. Five-day-old seedlings of Dro1-NIL were treated with 10 μM 2,4-D and/or 10 μM CHX. Data are means ± s.d.; n = 3 biological repeats (15 seedlings per repeat). P values are based on Student’s t-test.

DR

O1/

UB

Q (x

10-3

)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

2.5

3.0

Control 2,4-D CHX CHX

2,4-D +

P < 0.0001

P = 0.0003


a

0 -5 5

b

P = 0.1691

OsP

IN1a

(3 h

/0 h

ratio

)

0.0

1.0

2.0

3.0

4.0

IR64 Dro1-NIL

0.0

0.2

0.4

0.6

0.8

1.0

IR64 Dro1-NIL

OsP

IN2

(3 h

/0 h

ratio

) P = 0.6387

OsP

ID (3

h/0

h ra

tio)

0.0

1.0

2.0

3.0

4.0

5.0

IR64 Dro1-NIL

P = 0.439

Supplementary Figure 14. Expression profiling of auxin-related genes in IR64 and Dro1-NIL roots at 3 h after auxin treatment. (a) Microarray analysis based on a two-color system with auxin-treated samples (3 h; Cy5) and pre-treated samples (0 h; Cy3) was performed. FDR, false discovery rate. The log ratio of signal intensity (log2 Cy5/Cy3) was used to construct the heat map. The criterion for inclusion was FDR < 0.05 for at least one of the two samples (IR64 and Dro1-NIL). The data are means of three independent biological replicates, and the values for genes represented by multiple probes were averaged. Green vertical bar, auxin biosynthesis genes46; orange, signaling genes46; blue, polar transport genes28–30,47. The ARF protein used in the electrophoretic mobility-shift assay is OsARF23. (b) Quantitative RT-PCR analysis of auxin polar transport gene expression in response to auxin treatment. Expression data for OsPIN1a, OsPIN2, and OsPID were normalized to expression of a ubiquitin gene. P values are based on Student’s t-test. (a, b) Seedling root tips of IR64 and Dro1-NIL were treated with 10 μM 2,4-D for 0 or 3 h. Data are mean ± s.d.; n = 3 biological repeats (15 seedlings per repeat).

Gene name Locus IR64 Dro1-NIL IR64 Dro1-NIL IR64 Dro1-NIL

DRO1 Os09g0439800 -1.6 -2.1 0.021 0.010 0.002 0.002

OsASA2 Os03g0264400 0.6 0.6 0.035 0.009 0.011 0.001 OsASB1 Os04g0463500 0.6 0.6 0.035 0.014 0.012 0.004 OsTAA1;1 Os01g0169800 0.7 0.4 0.047 0.009 0.018 0.001 OsYUCCA7 Os04g0128900 -0.4 -0.8 0.037 0.010 0.011 0.002

OsARF1 Os01g0236300 -0.7 -0.6 0.057 0.008 0.025 0.000 OsARF4 Os01g0927600 -0.4 -0.4 0.053 0.018 0.024 0.008 OsARF6 Os02g0164900 0.9 1.0 0.077 0.021 0.047 0.011 OsARF7 Os02g0557200 -0.5 -0.5 0.056 0.009 0.025 0.001 OsARF9 Os04g0442000 0.2 0.3 0.256 0.017 0.199 0.007 OsARF10 Os04g0519700 1.5 2.1 0.035 0.010 0.010 0.001 OsARF11 Os04g0664400 2.0 1.8 0.029 0.010 0.007 0.001 OsARF12 Os04g0671900 1.0 1.2 0.023 0.012 0.004 0.004 OsARF14 Os05g0515400 1.4 1.6 0.032 0.010 0.009 0.002 OsARF15 Os05g0563400 1.7 2.0 0.045 0.016 0.017 0.006 OsARF17 Os06g0677800 0.4 0.4 0.078 0.021 0.042 0.010 OsARF18 Os06g0685700 -0.8 -0.7 0.016 0.010 0.001 0.002 OsARF19 Os06g0702600 0.2 0.5 0.497 0.049 0.442 0.035 OsARF23 Os11g0523800 1.0 0.8 0.058 0.011 0.026 0.003 OsARF24 Os12g0479400 -0.6 -0.5 0.042 0.010 0.016 0.002 OsARF25 Os12g0613700 -0.3 -0.3 0.073 0.023 0.039 0.012 OsIAA1 Os01g0178500 2.0 2.1 0.024 0.010 0.004 0.001 OsIAA2 Os01g0190300 0.5 0.7 0.167 0.015 0.118 0.006 OsIAA4 Os01g0286900 3.1 3.4 0.026 0.008 0.005 0.000 OsIAA5 Os01g0675700 0.9 0.9 0.022 0.008 0.003 0.000 OsIAA6 Os01g0741900 0.8 1.0 0.070 0.009 0.035 0.001 OsIAA9 Os02g0805100 4.6 4.8 0.021 0.009 0.003 0.001 OsIAA10 Os02g0817600 0.9 1.1 0.038 0.021 0.013 0.010 OsIAA11 Os03g0633500 0.7 0.6 0.043 0.018 0.016 0.008 OsIAA12 Os03g0633800 0.9 1.0 0.022 0.010 0.003 0.001 OsIAA13 Os03g0742900 0.8 0.8 0.017 0.010 0.001 0.002 OsIAA14 Os03g0797800 0.9 1.3 0.233 0.010 0.177 0.002 OsIAA15 Os05g0178600 2.3 2.1 0.022 0.008 0.003 0.000 OsIAA16 Os05g0186900 3.1 3.2 0.024 0.011 0.004 0.002 OsIAA20 Os06g0166500 7.2 6.4 0.019 0.009 0.002 0.001 OsIAA21 Os06g0335500 1.5 1.7 0.071 0.022 0.036 0.011 OsIAA22 Os06g0355300 -1.1 -0.9 0.032 0.012 0.009 0.003 OsIAA23 Os06g0597000 1.8 1.7 0.017 0.011 0.001 0.003 OsIAA24 Os07g0182400 1.4 1.6 0.023 0.009 0.004 0.001 OsIAA26 Os09g0527700 2.5 2.5 0.021 0.011 0.003 0.002 OsIAA27 Os11g0221000 0.9 0.9 0.027 0.011 0.006 0.003 OsIAA30 Os12g0601300 0.5 0.4 0.037 0.015 0.012 0.006 OsTIR1;2 Os02g0759700 -0.5 -0.5 0.050 0.030 0.022 0.018 OsTIR1;5 Os11g0515500 0.9 0.8 0.025 0.014 0.005 0.005

OsPIN1a Os06g0232300 1.8 2.2 0.016 0.013 0.001 0.004 OsPIN1b Os02g0743400 0.9 1.3 0.097 0.029 0.057 0.017 OsPIN1c Os11g0137000 1.1 1.2 0.041 0.014 0.014 0.005 OsPIN1d Os12g0133800 1.1 1.3 0.060 0.019 0.027 0.009 OsPIN2 Os06g0660200 -0.6 -0.6 0.038 0.027 0.013 0.015 OsPIN8 Os01g0715600 3.1 3.7 0.041 0.012 0.014 0.004 OsGNOM1/CRL4 Os03g0666100 -0.2 -0.2 0.089 0.018 0.050 0.008 OsPID Os12g0614600 2.3 2.6 0.020 0.010 0.002 0.002

Cy5/Cy3 ratio (log2) FDR p


DRO1(a)/90min/IR64 g

DRO1(a)/90min/Dro1-NIL g

(g)

(i) (h) (k)

(m) (l)

Supplementary Figure 15. Expression patterns of DRO1 and histone H4 gene in tissues of root tips of IR64 and Dro1-NIL after horizontal rotation. (a–f) Longitudinal sections; (g–p) transverse sections. (a, c, e, g–j, o) Dro1-NIL; (b, d, f, k–n, p) IR64. DRO1(a), in situ hybridization with an antisense probe of DRO1; DRO1(s), in situ hybridization with a sense probe of DRO1; H4a(a), in situ hybridization with an antisense probe of the histone H4 gene as an S-phase marker gene to estimate the region of root apical meristem. Arrowheads with letters in parentheses in (a) and (b) indicate the positions of the transverse sections in (g–p). Arrowheads labeled h, l, o, and p indicate the same position as the arrowhead shown in Figure 4c. Black arrows indicate direction of gravitational force after rotation. “60min” and “90min” indicate roots sampled 60 min and 90 min after horizontal rotation, respectively. Scale bars, 200 μm.

Distal elongation zone (DEZ)

H4a(a)/90min/Dro1-NIL H4a(a)/90min/IR64 e f

g g

Root apical meristem Elongation zone

j

g

n

h DRO1(s)/90min/Dro1-NIL

g

p DRO1(a)/60min/IR64

g

a b

DRO1(s)/90min/Dro1-NIL DRO1(s)/90min/IR64

c d

g g

(o) (p)

i g

m l k

o

(j) (n)

DRO1(a)/90min/Dro1-NIL DRO1(a)/90min/Dro1-NIL DRO1(a)/90min/Dro1-NIL

DRO1(a)/90min/IR64 DRO1(a)/90min/IR64 DRO1(a)/90min/IR64 DRO1(a)/90min/IR64

g g

DRO1(a)/60min/Dro1-NIL

g

g g g g


0.000 0.005 0.010 0.015

0.000 0.005 0.010 0.015 0.020 0.025

0.000 0.005 0.010 0.015 0.020 0.025

0.000 0.005 0.010 0.015 0.020 0.025

Supplementary Figure 16. Expression of DRO1 and OsIAA20 after horizontal rotation around the distal elongation zone after horizontal rotation. (a) Same image as in Figure 4c, with sampling regions shown. Dashed areas indicate regions sampled for quantitative RT-PCR. Vertical arrow indicates direction of gravitational force after rotation. Scale bar, 500 μm. (b) Root tips of Dro1-NIL and IR64 seedlings were rotated 90º from the original vertical axis and sampled as shown in (a). Expression data for DRO1 and OsIAA20 were normalized relative to expression of a ubiquitin gene (UBQ). Data are means ± s.d.; n = 3 biological repeats (6 to 8 seedlings per repeat). P values are based on Student’s t-test.

1.5

1.0

0.5

Tim

e af

ter r

otat

ion

treat

men

t (h)

P = 0.0002

P = 0.0049

P = 0.0593

DRO1/UBQ in Dro1-NIL

P = 0.0980

P = 0.1365

P = 0.2715

OsIAA20/UBQ in Dro1-NIL

b

a

DRO1/UBQ in IR64

OsIAA20/UBQ in IR64

1.5

1.0

0.5

Tim

e af

ter r

otat

ion

treat

men

t (h)

1.5

1.0

0.5

Tim

e af

ter r

otat

ion

treat

men

t (h)

1.5

1.0

0.5

Tim

e af

ter r

otat

ion

treat

men

t (h)

P = 0.0091

P = 0.0478

P = 0.0337

P = 0.0220

P = 0.1330

P = 0.0540

Lower Upper

Upper g

Distal elongation zone (DEZ)

Root apical meristem Elongation zone


Upper

Lower

0 10 20 30 40 50 60 70 80 90

NIL

IR64 P = 0.8859

P = 0.0004

0 10 20 30 40 50 60 70 80 90

NIL

IR64 P = 0.3225

P = 0.0261

0 10 20 30 40 50 60 70 80 90

NIL

IR64 P = 0.0324

P = 0.0999

P = 0.8296

P = 0.0923

0 5 10 15 20 25

NIL

IR64

0 5 10 15 20 25

NIL

IR64

0 5 10 15 20 25

NIL

IR64 P = 0.4684

P = 0.3199

P = 0.1329

P = 0.3909

0 5 10 15 20 25

NIL

IR64

0 5 10 15 20 25

NIL

IR64

0 5 10 15 20 25

NIL

IR64

P = 0.7108

P = 0.7339

P = 0.3397

P = 0.5062

P = 0.1696

P = 0.6191

Cell number in sclerenchyma

Cell number in exodermis

Cell number in epidermis

Cell height in sclerenchyma (μm)

Cell height in exodermis (μm)

Cell height in epidermis (μm)

Cell width in sclerenchyma (μm)

Cell width in exodermis (μm)

Cell width in epidermis (μm)

0 50 100 150 200 250

NIL

IR64

Root radius (μm)

P = 0.1768

P = 0.6703

Lower Upper

Supplementary Figure 17. Comparisons of cell size and cell number in sclerenchyma, exodermis, and epidermis between upper and lower semicircles of transverse sections in root tips. (a) Image of measurement regions. Dashed line crossing through the center of the protoxylem indicates where transverse sections were separated into upper and lower sections with respect to gravitational force after rotation. Vertical and horizontal sides of each cell relative to center of root were designated as cell height and width. Arrow indicates direction of gravitational force after rotation. (b) Transverse sections were obtained in 600–800 μm from the quiescent center (around arrowhead with (i, j) in the Supplemental Fig. 15a) at 1.5 h after rotation. Data are means ± s.d.; n = 21 plants. Averages of cell length and width in each plant were obtained from all cells in each side. P values are based on Student’s t-test. NIL, Dro1-NIL.

b

a

g


P < 0.0001

Supplementary Figure 18. Effect of different levels of drought stress on shoot and yield-related traits measured in IR64 and Dro1-NIL. Data are means ± s.d. P values are based on ANOVA. Days to 50% flowering was assessed by using all of the plants of each genotype. NIL, Dro1-NIL.

IR64 NIL

No drought

Moderate drought

Severe drought

IR64 NIL IR64 NIL

Day

s to

50%

flow

erin

g

0

10

20

30

40

50

60

70

80

90

100

Plan

t hei

ght (

cm)

0

10

20

30

40

50

60

70

80

90

Num

ber o

f pan

icle

s pe

r pla

nt

0

5

10

15

20

25

Num

ber o

f spi

kele

ts p

er p

anic

le

0

20

40

60

80

100

120 N

umbe

r of f

illed

grai

ns p

er p

anic

le

0

10

20

30

40

50

60

70

80

90

0

10

20

30

40

50

60

70

80

90

Tota

l abo

ve-g

roun

d dr

y w

eigh

t (g)

P = 0.8388 P = 0.1500

P = 0.1566 P = 0.8160 P = 0.6716

P < 0.0001 P = 0.9694

P = 0.7104

P < 0.0001

P < 0.0001

P = 0.0126

P = 0.0002

P = 0.7678 P = 0.5254

IR64 NIL IR64 NIL

Moderate drought

Severe drought

IR64 NIL

No drought

(n = 24) (n = 23) (n = 23) (n = 23) (n = 21) (n = 24) IR64 NIL IR64 NIL

Moderate drought

Severe drought

IR64 NIL

No drought

(n = 24) (n = 23) (n = 23) (n = 23) (n = 21) (n = 24)

IR64 NIL IR64 NIL

Moderate drought

Severe drought

IR64 NIL

No drought


Moderate drought

Severe drought

IR64 NIL

No drought


Moderate drought

Severe drought

IR64 NIL

No drought

(n = 24) (n = 23) (n = 23) (n = 23) (n = 21) (n = 24)


Supplementary Figure 19. Time course of soil water potential at a depth of 40 cm in an upland field (measured at around 13:00 each day). To compare drought conditions between this drought experiment (at NIAS) and the first drought experiment (under a rainout shelter at CIAT; Figure 5), we estimated soil water potential (MPa) at a depth of 40 cm in the first experiment from the soil moisture (%) and soil texture at CIAT by using RETC v. 6.02 software (http://www.pc-progress.com/en/Default.aspx?retc-downloads), which is based on the model of van Genuchten53. The mean estimated soil water potential at a depth of 40 cm in the severe-drought plot at CIAT from 20 to 30 days after drought stress was around –0.035 MPa. Therefore, the drought stress under the experimental conditions shown here was more severe than in the severe-drought plot at CIAT.

Days after sowing

Re-irrigation Stopping of

irrigation

Soil

wat

er p

oten

tial (

MPa

)

-0.10

-0.09

-0.08

-0.07

-0.06

-0.05

-0.04

-0.03

-0.02

-0.01

0.00

80 90 100 110 120 130 140 150 160 170

Harvest time


Supplementary Figure 20. Physiological and morphological differences between IR64 and Dro1-NIL under drought stress conditions. (a) Visible and (b) thermal images of IR64 and Dro1-NIL at 30 days after the start of drought stress (field conditions are shown in Supplementary Figure 19). The scale at the right of b indicates the relationship between image color and temperature (ºC). The leaf temperature of Dro1-NIL was clearly lower than that of IR64. (c) Side and (d) top views of the block at 37 days after the start of drought stress. By then, all plants of Dro1-NIL showed panicle emergence. In IR64, few heading plants were observed and leaf rolling had occurred.

IR64 Dro1-NIL IR64 Dro1-NIL

a

b d

IR64 Dro1-NIL IR64 Dro1-NIL

c


P = 0.0009

P = 0.0020

P = 0.3724

P = 0.0007

P = 0.0010

P = 0.0196

P < 0.0001

P = 0.3996

P = 0.0011

P < 0.0001

P = 0.0404

P < 0.0001

Supplementary Figure 21. Effect of DRO1 on photosynthesis, yield performance, and vertical root distribution under drought stress. (a) Difference between canopy temperature of Dro1-NIL and IR64 (ºC) at 30 and 37 days after start of drought stress, indicating that the Dro1-NIL canopy was cooler. Data are from field experiment shown in Supplementary Figures 19 and 20. Data are means ± s.d.; n = 3 blocks. (b) Photosynthesis rate. Data are means ± s.d.; n = 9 plants (3 plants per block × 3 blocks). (c) Stomatal conductance. Data are means ± s.d.; n = 9 plants (3 plants per block × 3 blocks). (d) Days to 50% flowering in each block. Data are means ± s.d.; n = 3 blocks. (e) Total above-ground dry weight per plant. (f) Number of panicles per plant. (g) Grain weight per plant. (h) Grain weight per panicle. Data are means ± s.d.; n = 3 blocks (24 plants per block). (i) 1000-grain weight. Data are means ± s.d.; n = 9 plants (3 plants per block × 3 blocks). (j, k) Total root dry weight of A–L blocks per plant (j) and root dry weight for each soil block (k) obtained from soil monolith sampling. Data are means ± s.d.; n = 9 plants (3 plants per block × 3 blocks). Letters A–L in k indicate positions of soil blocks as described in Supplementary Figure 2d. Data are means ± s.d. P values are based on ANOVA.

0

2

4

6

8

10

12

14

IR64 Dro1-NIL

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

IR64 Dro1-NIL

0

5

10

15

20

25

30

IR64 Dro1-NIL

Gra

in w

eigh

t per

pla

nt (g

)

Gra

in w

eigh

t per

pan

icle

(g)

1000

-gra

in w

eigh

t (g)

i h g

Tota

l roo

t dry

wei

ght (

g)

j

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

IR64 Dro1-NIL

P = 0.1547 P = 0.0007 P = 0.0143 P = 0.0126

f

0 2 4 6 8

10 12 14 16 18 20

IR64 Dro1-NIL

Num

ber o

f pan

icle

s pe

r pla

nt

P = 0.0035

H

G

F

E

k

Root dry weight (g)

L

K

J

I

0.00 0.05 0.10 0.15

Root dry weight (g)

IR64 Dro1-NIL

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Root dry weight (g)

D

C

B

A

0.00 0.05 0.10 0.15

Roo

t pos

ition

in

soil

mon

olith

blo

ck

Days after start of stress

Diff

eren

ce b

etw

een

cano

py te

mpe

ratu

re

of D

ro1-

NIL

and

IR64

(ºC

)

-1.8

-1.6

-1.4

-1.2

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

30 37

a e

0

10

20

30

40

50

60

70

IR64 Dro1-NIL

Tota

l abo

ve-g

roun

d dr

y w

eigh

t pe

r pla

nt (g

)

P = 0.1830

100

105

110

115

120

125

IR64 Dro1-NIL

Day

s to

50%

flow

erin

g

d

P = 0.0058

b

0 2 4 6 8

10 12 14 16 18 20

IR64 Dro1-NIL

Phot

osyn

thes

is ra

te

(μm

ol C

O2 m

-2 s

-1)

P < 0.0001

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Stom

atal

con

duct

ance

(m

ol m

-2 s

-1)

IR64 Dro1-NIL

P = 0.0004

c


Drought plot

25 cm

5 cm Gravel layer

No-drought Irrigated plot

Bed soil

a b

Soil

wat

er p

oten

tial (

MPa

)

Days after sowing

Tensiometer

-0.10 -0.09 -0.08 -0.07

-0.06 -0.05

-0.04 -0.03

-0.02 -0.01 0.00

35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150

c

Re-irrigation Stopping of

irrigation Harvest time

Supplementary Figure 22. Experimental conditions in an artificial drought stress system in which access to soil water is limited. (a) Cross-sectional diagram of physical environments in drought avoidance assay. Blue arrows represent capillary water flow in the soil. Blue dashed lines indicate plastic nets between gravel and soil layers. (b) View of assay system in the greenhouse before the start of drought stress. (c) Time course of soil water potential in the artificial drought conditions (measured at around 13:00 each day).

25 cm below ground (No drought) 40 cm below ground (No drought) 25 cm below ground (Drought) 40 cm below ground (Drought)

Upper soil layer


Supplementary Figure 23. Effect of DRO1 on drought avoidance in an artificial drought system in which access to soil water is limited. (a) Cross-sections of assay system (top panels) and views from above gravel layer (bottom panels) in the drought plot at harvest time. Yellow asterisks in upper right image indicate roots that had elongated through the gravel layer. Yellow arrowheads in lower right image indicate roots penetrating into the gravel layer. Some roots of Dro1-NIL, but not IR64, grew through the gravel layer. (b) Top views of rice plants at 28 to 49 days after the start of drought stress. At later stages, many IR64 plants in the drought plot had leaf rolling, and some had leaf wilting. (c) Effect of drought stress on shoot- and yield-related traits measured in IR64 and Dro1-NIL. Data are means ± s.d.; n = 16 plants. P values are based on ANOVA.

b IR64

Dro1-NIL

Dro1-NIL

IR64

28 35 42 49 Days after start of stress

No-drought plot

Drought plot

a

IR64 Dro1-NIL

* * *

* * * *

*

*

* *

Gravel layer

c

Cul

m le

ngth

(cm

)

0

10

20

30

40

50

60

70

No Drought Drought

IR64 Dro1-NIL

Tota

l abo

ve-g

roun

d dr

y w

eigh

t (g)

0

20

40

60

80

100

120

140

160

Drought No Drought

Pani

cle

leng

th (c

m)

0

5

10

15

20

25

30

Drought No Drought

Gra

in w

eigh

t per

pla

nt (g

)

0

2

4

6

8

10

12

Drought No Drought

P = 0.2495

P = 0.7339

P = 0.7684

P = 0.9351

P = 0.1033

P = 0.0256

P = 0.5248

P < 0.0001


IR64

Dro1-NIL

Kinandang Patong

PEG concentration

0%

1

1

1

10%

1

1

1

30%

9

9

9

20%

3

3

5

Supplementary Figure 24. Comparison of dehydration tolerance of IR64, Dro1-NIL, and Kinandang Patong seedlings grown in hydroponic culture with polyethylene glycol (PEG). Response to osmotic stress of rice plants at 2 days after PEG treatment. Eight-day-old seedlings were transferred to a PEG solution (0%–30%). The number in each photo indicates the dehydration-tolerance score on a scale of 1 (tolerant of osmotic stress) to 9 (intolerant). In the 20% PEG treatment, the young leaves of Kinandang Patong were tightly rolled, whereas those of IR64 and Dro1-NIL were curved but not rolled. In the 30% PEG treatment, all plants showed leaf wilting. Consistent with the results shown here, IR64 was shown to be more tolerant to dehydration than Kinandang Patong in a previous study using other drought treatment54.


5' UTR 5' UTR Exon 3 Exon 3 Exon 4 Exon 4 3' UTR A/G C/T T/C A/C A/- C/A CT/--

S.S. Leu/Phe Stop Asp/Gly 27a 150a 617a 752a 943a 962a 1133-4a

SNP SNP SNP SNP In/del SNP In/del WRC55 Tupa 729 Bangladesh japonica A C T A A C CT I WRC53 Tima Bhutan japonica A C T A A C CT I WRC47 Jaguary Brazil japonica A C T A A C CT I WRC52 Khau Tan Chiem Vietnam japonica A C T A A C CT I WRC21 Shwe Nang Gyi Myanmar indica A C T A A C CT I WRC42 Local Basmati India indica A C T A A C CT I WRC67 Phulba India japonica A C T A A C CT I WRC68 Khao Nam Jen Laos japonica A C T A A C CT I WRC1 Nipponbare Japan japonica A C T A A C CT I WRC43 Dianyu 1 China japonica A C T A A C CT I WRC45 Ma sho Myanmar japonica A C T A A C CT I WRC50 Rexmont USA japonica A C T A A C CT I WRC46 Khau Mac Kho Vietnam japonica A C T A A C CT I WRC44 Basilanon Philippines indica A C T A A C CT I WRC51 Urasan 1 Japan japonica A C T A A C CT I WRC49 Padi Perak Indonesia japonica A C T A A C CT I IRGC23364 Kinandang Patong Philippines japonica A C T A A C CT I WRC35 ARC 5955 India indica A T C A A C - II WRC2 Kasalath India indica A T C A A C - II WRC13 Asu Bhutan indica A T C A A C - II WRC30 Anjana Dhan Nepal indica A T C A A C - II WRC41 Kaluheenati Sri Lanka indica A T C A A C - II WRC39 Badari Dhan Nepal indica A T C A A C - II WRC40 Nepal 555 India indica A T C A A C - II WRC29 Kalo Dhan Nepal indica A T C A A C - II WRC25 Muha Indonesia indica A T C A A C - II WRC26 Jhona 2 India indica A T C A A C - II WRC38 ARC 11094 India indica A T C A A C - II WRC27 Nepal 8 Nepal indica A T C A A C - II WRC36 Ratul India indica A T C A A C - II WRC9 Ryou Suisan Koumai China indica A T C A A C - II WRC33 Surjamukhi India indica A T C A A C - II WRC31 Shoni Bangladesh indica A T C A A C - II WRC97 Chin Galay Myanmar indica A T C C A C - III WRC96 Pokkari India indica A T C C A C - III WRC6 Puluik Arang Indonesia indica A T C C A C - III WRC62 Kemasin Malaysia indica A T C C A C - III WRC3 Bei Khe Cambodia indica A T C C A C - III WRC61 Radin Goi Sesat Malaysia indica A T C C A C - III WRC64 Padi Kuning India indica A T C C A C - III WRC65 Rambhog India indica A T C C A C - III WRC14 IR 58 Philippines indica A T C C A C - III WRC4 Jena 035 Nepal indica A T C C A C - III WRC5 Naba India indica A T C C A C - III WRC58 Neang Menh Cambodia indica A T C C A C - III WRC16 Vary Futsi Madagascar indica A T C C A C - III WRC57 Milyang 23 Korea indica A T C C A C - III WRC28 Jarjan Bhutan indica A T C C A C - III WRC100 Vandaran LKA indica A T C C A C - III WRC34 ARC 7291 India indica A T C C A C - III WRC24 Pinulupot 1 Philippines indica A T C A A A - IV WRC99 Hong Cheuh Zai China indica A T C A A A - IV WRC17 Keiboba China indica A T C A A A - IV WRC10 Shuusoushu China indica A T C A A A - IV WRC18 Qingyu (Seiyu) China indica A T C A A A - IV WRC19 Deng Pao Zhai China indica A T C A A A - IV WRC98 Deejiaohualuo China indica A T C A A A - IV WRC66 Bingala Myanmar indica A T C A A A - IV WRC7 Davao 1 Philippines indica A T C A A A - IV WRC20 Tadukan Philippines indica A T C A A A - IV WRC63 Bleiyo Thailand indica G T C A A C - V WRC11 Jinguoin China indica G T C A A C - V WRC60 Hakphaynhay Laos indica G T C A A C - V IRGC66970 IR64 Philippines indica G T C A - C - VI

Variety name Origin Variety group

Sequence variation in transcribed region of DRO1

Haplotype group

Accession No.

Ratio of deep rooting (%) 0 20 40 60 80 100

Supplementary Figure 25. Sequence polymorphism in transcribed region of DRO1 and natural variation in deep rooting among cultivars of cultivated rice. WRC, National Institute of Agrobiological Sciences; IRGC, International Rice Research Institute. S.S., synonymous substitution. a In/del or SNP position from start of the 5′ UTR. Rooting data are means ± s.d.; n = 5 plants.


Supplementary Figure 26. Sequence polymorphism of In/del in exon 4 of DRO1 and natural variation in deep rooting among accession lines related to IR64. JP, National Institute of Agrobiological Sciences; IRGC, International Rice Research Institute. Rooting data are means ± s.d.; n = 5 plants.

Accession No. Variety name Species Origin In/del (943 bp) IRGC 39287 IR 2061-465-1-4 O. sativa Philippines A JP 12462 IR 2061-465-1-5-5 O. sativa Philippines A IRGC 831 GAM PAI 30-12-15 O. sativa Thailand A IRGC 32662 IR 1833 O. sativa Philippines A IRGC 32645 IR 1737 O. sativa Philippines A IRGC 32644 IR 1721 O. sativa Philippines A JP 12495 IR 400-28-4-5 O. sativa Philippines A JP 80814 TE-TEP O. sativa Philippines A IRGC 32641 IR 1704 O. sativa Philippines A JP 12435 IR 262-43-8-11 O. sativa Philippines A IRGC 32629 IR 1614 O. sativa Philippines A JP 12438 IR 747-B2-6-3 O. sativa Philippines A JP 14213 IR 579-48-1-2 O. sativa Philippines A JP 12469 IR22 O. sativa Philippines A IRGC 16319 IR 1006 O. sativa Philippines A IRGC 5070 CHOU SUNG O. sativa South Korea A JP 12436 IR24 O. sativa Philippines A IRGC 101508 - O. nivara India A IRGC 11374 IR 773 A 1-36-2-1 O. sativa Philippines A JP 12485 TADUKAN O. sativa Philippines A JP 12467 IR8 O. sativa Philippines A JP 12483 BPI 76 (BICOL STRAIN) O. sativa Philippines A JP 12491 MUDGO O. sativa Philippines A IRGC 15762 BPI 121-407 O. sativa Philippines A JP 12331 TAICHUNG NATIVE 1 O. sativa Taiwan A JP 81859 TKM 6 O. sativa India A JP 84630 DEE GEO WOO GEN O. sativa Taiwan A IRGC 14468 SERAUP BESAR 15 O. sativa Malaysia A JP 14720 FORTUNA O. sativa USA A JP 12812 SIGADIS O. sativa Indonesia A JP 12473 B 5580 AL 15 O. sativa Philippines A IRGC 172 NANHNG MON S 4 O. sativa Thailand A JP 84627 TSAI YUAN CHUNG O. sativa Taiwan A JP 14727 BLUE BONNET O. sativa USA A JP 15075 CENTURY PATNA 231 O. sativa USA A JP 13041 GEB 24 O. sativa India A JP 14690 TEXAS PATNA O. sativa USA A JP 14735 REXORO O. sativa USA A IRGC 117410 IR07F102 O. sativa Philippines - IRGC 117405 IR 64-21 (SALINITY SELECTION) O. sativa Philippines - IRGC 117387 NSIC RC 140 (TUBIGAN 6) O. sativa Philippines - IRGC 99662 IR 64683-87-2-2-3-3 O. sativa Philippines A IRGC 120987 IR 77298-14-1-2 O. sativa Philippines A IRGC 117297 IR 75870-5-8-5-B-1 O. sativa Philippines A IRGC 117374 IR 77298-14-1-2 O. sativa Philippines A IRGC 115997 IR 72757-529-B-B O. sativa Philippines A IRGC 117292 IR 73678-20-1-B O. sativa Philippines A IRGC 116797 IR 84629-10 MP O. sativa Philippines A

Parental lines of IR64

Progeny lines of IR64

0 20 40 60 80 100

Ratio of deep rooting (%)


Supplementary Table 1. Sequence polymorphism of In/del in exon 4 of DRO1 among wild relatives of rice.

Accession No. Species Origin In/del (943 bp)

AS13 O. nivara India A

AS33 O. nivara Thailand A AS34 O. nivara Thailand A AS37 O. nivara Thailand A


AS41 O. nivara Thailand A AS44 O. nivara Thailand A

AS49 O. nivara Sri Lanka A AS50 O. nivara Nepal A AS51 O. nivara Nepal A

AS57 O. nivara Thailand A


AS19 O. rufipogon Thailand A AS21 O. rufipogon Thailand A AS23 O. rufipogon Thailand A AS31 O. rufipogon Thailand A AS32 O. rufipogon Thailand A AS39 O. rufipogon Thailand A AS66 O. rufipogon Philippines A AS72 O. rufipogon India A AS73 O. rufipogon India A AS74 O. rufipogon India A AS75 O. rufipogon Laos A AS76 O. rufipogon Vietnam A AS77 O. rufipogon Papua New Guinea A AS78 O. rufipogon Cambodia A AS79 O. rufipogon Myanmar A AS46 O. meridionalis Australia A

The accession numbers of AS are from the National Institute of Agrobiological Sciences.


Supplementary Table 2. Primer pairs used for high-resolution mapping, sequence analysis, and electrophoretic mobility-shift assays (EMSAs), and for making the RNAi and CaMV35S::DRO1::YFP constructs and the in situ hybridization probe.

CAPS, cleaved amplified polymorphic sequence; In/del, insertion/deletion mutation; SSR, simple sequence repeat.

Primer name Forward primer (5'-3') Reverse primer (5'-3') Purpose

RM24393 TAGCTGCTTAGCTTTGACTTGG ATGTAATCCTACGAGGAGATCG SSR marker

RM7424 CAGATCAAGCTAGCCACACAGC GAAGGCAGAGCAGGAGAGAAGC SSR marker

CAPS05 GCACAAGATGGGAGGAGAGT CATGGGTGAGAATCGTGTTG CAPS marker (HinfI)

INDEL09 GCAGACGCTCGTAACACGTA GTGGCAGCTCCATCAACTCT In/del marker

Dro1seq-00 ATATGGGCGTACGGTAGCTG AGAGATTGGGGAGGGAGAAA Sequence analysis

Dro1seq-01 GCTGTGTCCTGTTATCATTCCA CCTCAAGGAACAGGGAAACA Sequence analysis

Dro1seq-02 CTTGCGGCTTAATCGAGTTC GGAAGAATTTTGCGGGTGTA Sequence analysis

Dro1seq-03 AGGGAGTGGAGTAAGCATGG AGCAACGAAGCGACTGATCT Sequence analysis

Dro1seq-04 TGCCACTTTTGTCAATGGAG TGCCCGTACTGTACCAACAA Sequence analysis

Dro1seq-05 AGGGAGTGGAGTAAGCATGG ATCGGCACGCTTTTGTAAAC Sequence analysis

Dro1seq-06 GTAAGCATGGGCAGACATTG ATCGGCACGCTTTTGTAAAC Sequence analysis

Dro1seq-07 TGAAAACATCAGGGAGTGGA ATCGGCACGCTTTTGTAAAC Sequence analysis

Dro1seq-08 GACGATGATGGTGCAAAATG CCTTTGTCCCAGAACCTCCT Sequence analysis

Dro1seq-09 GACGATGATGGTGCAAAATG GGCAGACAACTCTGGAATCA Sequence analysis

Dro1seq-10 GGTGCAAAATGGGTCAAAAC GGCAGACAACTCTGGAATCA Sequence analysis

Dro1-In/del(943) AGATCAGTCGCTTCGTTGCT ACCTGGCATGAACGAACTAA Sequence analysis

Dro1-trigger01 CACCCATTGCTTGCTATTGGGACA AGCAACGAAGCGACTGATCT RNAi construct

Dinsitu-P TTCATCCGACAATGTGCAGT GAACCTGGCATGAACGAACT In situ hybridization

Dro1-kp(cDNA) CACCATGAAGATTTTCAGCTGGGT CATTTCAAGCACAATATACT Subcellular localization

Frag.1 GGTATATCGAGATGATGGTT TGTACGATATTTCGAGACAC EMSA

Frag.2 TCCGAAGAAAGTGCAAATTC CAGGTGAGCCATGGAATGAT EMSA

Frag.3 GGAGTCATCTATAAAAGGAA GGCTGTAGGATAGCTTGCTA EMSA

Frag.1m GGTATATCGAGATGATGGTT TGTACGATATTTCGAGATAC EMSA

pCRII CCAGTGTGCTGGAATTCGGC GGATATCTGCAGAATTCGGC EMSA

Dro1-ir(cDNA) CACCATGAAGATTTTCAGCTGGGT GATCACGCAGTGCACCTCC Subcellular localization


Supplementary Table 3. Primer pairs and TaqMan probes used for quantitative RT-PCR.

Primer name

DRO1

OsIAA20

OsPIN1a

OsPIN2

OsPID

Ubiquitin

Forward primer (5'–3')

GCAAGAAGCAAATCGGTTTCC

CGGGATTATTTTGTTCACGTTTC

CCTGACAACCAGCCATGTTA

TGGGCTTTCTTGAAGACCTG

GGTTCGACCTCTTCTGAGCA

GAGCCTCTGTTCGTCAAGTA

Reverse primer (5'–3')

GAATTCATCCTTTCGACAATCTGA

CGAGATTTCATTCGTCATGCTTA

CTCGTTTGACTCCCTTCCAA

GATGCCAAAACAGTTGCAGA

CGGTAGCTAAGGGTCAACGA

ACTCGATGGTCCATTAAACC

TaqMan probe

ATTCTTCTGCGCCTTACCGTGCTAAC

–

–

–

–

TTGTGGTGCTGATGTCTACTTGTGTC


Supplementary Table 4. Summary of the traits evaluated in the field or greenhouse.

Figure no. Trait Sampling time Growth condition Country

Figure 1a Root distribution 120 days after sowing Upland condition with irrigation Japan

Figure 1c, d Ratio of deep rooting 35 to 42 days after sowing Basket with soil in greenhouse Japan

Figure 3g Ratio of deep rooting 35 to 42 days after sowing Basket with soil in greenhouse Japan

Figure 5c Filled grain percentage Grain weight per plant

After harvesting Upland condition with/without drought stress

Colombia

Supplementary Figure 1 Root distribution 120 days after sowing Upland condition with irrigation Japan

Supplementary Figure 3 All shoot and root traits 120 days after sowing Upland condition with irrigation Japan

Supplementary Figure 5 All shoot and root traits Temporal sampling Hydroponic condition in greenhouse Japan

Supplementary Figure 12 All floral traits Before flowering Paddy field Japan

Supplementary Figure 18 All shoot and yield-related traits

After harvesting Upland condition with drought stress Colombia

Supplementary Figure 21 Canopy temperature 30 or 37 days after start of stress

Upland condition with drought stress Japan

Photosynthesis rate 30 days after start of stress Upland condition with drought stress Japan Yield-related traits After harvesting Upland condition with drought stress Japan

Supplementary Figure 4 Root growth angle 35 days after sowing Basket with soil in greenhouse Japan

Supplementary Figure 23 All shoot and yield-related traits

After harvesting Upland condition with drought stress in greenhouse

Japan


control of root system architecture by deeper rooting 1 ......kp‒yfp (expected size 59.0 kda) and...

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