C

D

0

2

4

6

8

WT mutant

Wid

th o

f le

af b

lad

e (

mm

)

0

2

4

6

8

WT mutant

Wid

th o

f b

asa

l ste

m (

mm

)

E F

* *

A

Supplemental Figure S1. Phenotypic comparison of lateral roots, leaf blade and basal stem between

the wild-type rice (WT) and short-root mutant. A-B, lateral roots of WT (A) and mutant (B) grown for

7 days. C, leaf blade; D, basal stem; E-F, width of leaf blade (E) and basal stem (F) of WT (left) and

mutant (right) grown for 30 days. Error bars represent + SD (n=10). Scale bar =1 cm (A, B) and 2 cm

(C, D). The asterisk in (E) and (F) shows a significant difference between WT and mutant (P<0.05 by

Tukey’s test).

B

0

50

100

150

200

Nipponbare mutant

0

5

10

15

Nipponbare mutant

0

10

20

30

Nipponbare mutant

0

20

40

60

80

100

Nipponbare mutant

0

20

40

60

80

100

Nipponbare mutant

0

10

20

30

40

Nipponbare mutant

Supplemental Figure S2. Growth and grain yield of wild-type rice (cv. Nipponbare) and short-root mutant

grown in a field. A, Growth of Nipponbare (left) and mutant (right) grown in a paddy field. B, Plant height

of Nipponbare and mutant at harvest. C-D, Panicles (C) and seeds (D) of Nipponbare (left) and mutant

(right). Grain number = 50. E, Panicle number per plant. F, 1000-grain weight. G, Spikelet number per

panicle. H, Percentage of filled spikelet. I, Grain yield per plant. The wild-type rice and short-root mutant

were grown in a paddy field till ripening. Data are means + SD (n=12). The asterisk shows a significant

difference between WT and mutant (P<0.05 by Tukey’s test).

A

B E F

G H I

*

*

*

*

*

*

Heig

ht (c

m)

Pa

nic

le n

um

be

r p

lan

t-1

10

00

-gria

n w

eig

ht (g

)

% f

ille

d s

pik

ele

t

Gra

in y

ield

(g p

lan

t-1)

D

C S

pik

ele

t n

um

be

r p

an

icle

-1

0

2

4

6

8

10

Num

be

r o

f ro

ot co

rtic

al ce

ll la

ye

r

0

5

10

15

WT osccc1

Supplemental Figure S3. Comparison of cell number in roots and shoots between the wild-type

rice (WT) and short-root mutant. A, Number of cortical cell layer in the mature region (at 10 mm

from the apex) of the root (n=10). B, Number of adaxial epidermal cells between large and small

vascular bundles in leaf sheath (n=7).

Nu

mb

er

of

ad

axia

l e

pid

erm

al

ce

lls b

etw

ee

n v

ascu

lar

bu

nd

les

A

B

mutant

mutant WT

Marker 0811.4 0815.2 R8M23 0811.9

0814.4

0814.0 0814.5

Chr. 8

8 3 1 1 32 2 1

440 kb

0812.5

1

0814.2

0

Physical distance

n=3460

14,000K 14,100K 14,200K 14,300K 14,400K

Recombinants

Whole genome sequencing

Supplemental Figure S4. Map-based cloning of the gene responsible for the short-root phenotype.

A, Map-based cloning. Vertical lines represent molecular markers, and the numbers of recombinants

are indicated under vertical lines. The long horizontal line represents the rice chromosome 8. The

filled black oval in the long horizontal line represents centromere. The scale bar represents physical

distance. Using 3460 short-root plants (F2) from short-root mutant/Kasalath population, candidate

genes were mapped to a 440-kb region near the centromere of chromosome 8 (grey color). Further

using the whole genome sequencing, one point mutation was found at the position of 14,276,235 bp,

which was located in the gene of OsCCC1 (red box). B, Gene structure of OsCCC1. Grey boxes

indicate UTR regions, black boxes indicate exons, and black horizontal lines represent introns.

OsCCC1 contains 14 exons and 13 introns. The position of the mutation is indicated with a filled

grey triangle, which changes the nucleotide from G to T, and the amino acid from cysteine (C) to

phenylalanine (F).

WT mutant

TTT

(F)

TGT

(C)

ATG TGA

B

A

OsCCC1

Short-root mutant

Selfing

F2 progeny F1

Wild type

Short root (mutant)

Normal root (WT/hetero)

DNA sequencing & SNP

mapping

segregation

SNP index=17/44=0.39

A

A

C

A

A

A

C

C

CCAACA AAAGCA A C

.

. .

SNP index=49/49=1

A

A

A

A

A

A

A

A

CCAACA AAAGCA A

. . .

Wild type

Supplemental Figure S5. Scheme for MutMap using whole genome sequencing. The mutant plant

was crossed with the wild-type rice (Nipponbare). The F1 plant was self-pollinated to generate F2

progeny. The genomic DNA of F2 plants (n=50 each) showing the short-root or normal-root

phenotypes were bulked and subjected to whole genome sequencing.

Chr. 8 (14,276,235 bp)

Mutation position:

chr08:14276235 WT/He reads alignment results

Mutant reads alignment results chr08:14276235

CACAA

WT

CAMAA CAAAA CAMAA CAAAA

Mutant-1 Mutant-2 WT/He-1 WT/He-2

B

A

Supplemental Figure S6. Alignment of mutation region between the bulked DNA from normal-root and

short-root pools by MutMap (A) and confirmation of mutation by PCR (B). The point mutation locates at

14276235 bp in chromosome 8. The alignment region from 14276180 bp to 14276290 bp was shown in

(A). The number of reads covering the mutation locus in normal-root pool and short-root pool is 44 and

49, respectively in (A). 3 DNA samples from F2 population (short-root mutant/Nipponbare) showing the

short-root phenotype (Mutant-1 and Mutant-2) or normal-root phenotype (WT/He-1 and WT/He-2) were

equivalently mixed for sequencing of PCR products containing the mutation locus in (B). Mutant: short-

root phenotype; WT/He: normal-root phenotype.

F2 population

XP_002440935 (Sorghum bicolor)

XP_008656210 (Zea mays)

XP_008650609 (Zea mays)

OsCCC1 (Sativa Oryza)

OsCCC2 (Sativa Oryza)

XP_003533835 (Glycine max)

XP_003546564 (Glycine max)

AXI4 (Nicotiana tabacum)

CDX90250 (Brassica napus)

AtCCC1 (Arabidopsis thaliana)

CDY12056 (Brassica napus)

CDX77592 (Brassica napus)

CDX98204 (Brassica napus)

XP_002450297 (Sorghum bicolor)

XP_008680718 (Zea mays)

Supplemental Figure S7. Phylogenetic tree of OsCCC1 in plant (A) and predicated topology of

OsCCC1 (B). The star mark indicates the mutation site.

B

XP_003595505 (Medicago truncatula)

COOH

NH2

Cytosol

A

0

20

40

60

80

100

0 2 10 50

WT

osccc1

line 1

line 2

0

20

40

60

80

100

0 2 10 50

WT

osccc1

line 1

line 2

Roo

t le

ng

th (

mm

)

Roo

t le

ng

th (

mm

)

* *

* * * *

Supplemental Figure S8. Partial recovery of the root growth in short-root mutant by addition of

NaCl/KCl. Effect of the addition of different concentration of KCl (A) NaCl (B) on root growth.

Germinated seedlings were exposed to a solution containing KCl or NaCl (0, 2, 10, 50 mM) for 3 days.

The root length was measured by a ruler and was shown. Data are means + SD (n=10). The asterisk

indicates significant differences compared with WT (*P<0.05 by Tukey’s test).

A

KCl (mM) NaCl (mM)

B

mutant mutant

Distance from central cylinder (μm)

100 200

100

50

0

200

100

100 200 400 300 0

S H

100

50

100 200 400 300 0

S H

A B

E

H

K+

D

I

Na+ Cl-

Supplemental Figure S9. Elemental distribution in short-root mutant using SEM & EDS. Seedlings of both wild-type rice (A,

B, C, G, H, I) and mutant (D, E, F, J, K, L) were treated with 50 mM NaCl for 24 h. Root was excised and fixed by 5% agar

powder. The transverse section at 10 mm from the apex was used for analysis immediately. The scanning electro photos and

elemental distribution photos were generated by SEM and EDS, respectively. Pink color indicates K+ distribution (A, D). Red

color indicates Na+ distribution (B, E). Yellow color indicates Cl- distribution (C, F). Signal intensity of K+ (G, J), Na+ (H, K)

and Cl- (I, L) from stele (S) to exodermis (E) was quantified by EDS. Six root samples were investigated and showed a similar

tendency to the results presented in this figure. Bar= 200 μm.

C

F

G

Sig

na

l in

ten

sity

100

50

100 200 400 300

0

Sig

na

l in

ten

sity

J K L

S H

S H

S

H

H

S H

Distance from central cylinder (μm)

100 200

100

50

0 S H

Distance from central cylinder (μm)

100 200

200

100

0 S H

0

20

40

60

80

Ca(NO3)2 NaCl KCl

WT osccc1 line 1 line 2

Ca(NO3)2 KCl NaCl Ca(NO3)2 KCl NaCl

Root Shoot

*

*

*

*

*

*

K+ c

on

ce

ntr

ation (

mg

g-1

)

Supplemental Figure S10. Concentration of K+, Na+ and Cl- in roots and shoots in response to KCl and

NaCl. Germinated seedlings of wild-type rice (WT), short-root mutant and two independent

complementation lines were exposed to 0.1 mM Ca(NO3)2 solution with 0 mM NaCl/KCl, 50 mM KCl or

50 mM NaCl for 24h. Both shoots and roots were harvested for elemental analysis. The K+ (A) and Na+ (C)

concentrations were determined by ICP-MS. The Cl- (B) concentration was determined by ion

chromatograph. Data are means + SD (n=3). The asterisk indicates significant differences compared with

WT (*P<0.05 by Tukey’s test).

A

B

mutant

0

5

10

15

20

25

Ca(NO3)2 NaCl KCl

WT osccc1 line 1 line 2

*

*

Ca(NO3)2 KCl NaCl Ca(NO3)2 KCl NaCl

Root Shoot

Na

+ c

on

ce

ntr

ation

(m

g g

-1)

C

mutant

0

5

10

15

20

25

30

35

Ca(NO3)2 NaCl KCl

WT osccc1 line 1 line 2

*

* * *

*

Ca(NO3)2 KCl NaCl Ca(NO3)2 KCl NaCl

Root Shoot

Cl-

co

nce

ntr

ation

(m

g g

-1)

*

*

mutant

0

50

100

150

200

250

300

350

400

450

Ca(NO3)2 NaCl KCl

WT osccc1 line 1 line 2

Ca(NO3)2

*

*

*

Osm

ola

lity in s

ho

ot ce

ll (

mO

sm

Kg

-1)

Supplemental Figure S11. Osmolality in shoot cell sap in response to KCl and NaCl. Germinated

seedlings of wild-type rice (WT), short-root mutant and two independent complementation lines were

exposed to 0.1 mM Ca(NO3)2 solution with 0 mM NaCl/KCl, 2 mM KCl or 2 mM NaCl for 3d. The

shoot part was excised for cell sap collection. The osmolality was measured by vapor pressure

osmometer. Data are means + SD (n=3). The asterisk indicates significant differences compared with

WT (*P<0.05 by Tukey’s test).

mutant

250

150

100

75

50

37

25

20

15

10

Supplemental Figure S12. Western blot analysis of OsCCC1. Microsomal fraction of proteins was

extracted from roots and shoots of wild-type rice. An antibody against OsCCC1 or H+-ATPase was

used for western blot. H+-ATPase was used as an internal control.

OsCCC1

(108 kDa, predicted)

Root Shoot

100

75

H+-ATPase (90 kDa)

Supplemental Table S1. Primers for InDel markers used for mapping of OsCCC1

Primer Name Forward (5'---------3') Reverse (5'---------3')

0811.4 TACCAACGTCAATGTGCCGC TGGAAGATGGGCGTGAGGAA

0811.9 TTGCGATAACGGTGAAAGAG ACACGTGCGATTTTGCAAAC

0812.5 AGCGACGGCTAGGGTTTCTT AACCTGGGATGTTACATCTT

R8M23 CCTATTCACTCTACCGACAT GTTTAGTTCCCATTGCTTT

0814.0 AGAAAAGGAGGGAAACTGAA CGTCTAAGATTTCGATGAG

0814.2 AGTTGGAAACCTTTCCCT CCAAAATGCTAAACGGTGTG

0814.4 TAACACTAACTGGCAACC GATATAGAGGCTGGATTT

0814.5 ATTAAGGCAAGTTCTAAGCA CACCAATCAGCCATCTCATT

0815.2 CCTGAGGATTGTTATTGTCTC AACACCAATGAAGCAGAGCC