1

2

Supplemental Figure S1. Southern-blot linkage analysis of the pollen semi-sterile (PSS) 3

phenotype (cap1 mutation) and Tos17 insertions. Ten or more bands showing the Tos17 4

transposition were detected in each lane. An approximately 3-kb band (arrow) was always detected 5

in plants with the PSS phenotype (m lanes) but never in wild-type plants (w lanes). The leftmost 6

lane contained lambda DNA/HindIII marker; numbers are sizes. 7

1

2

3

Supplemental Figure S2. Genotyping of ND2104-line plants by PCR. The PCR mixture contained 4

three primers (CP-2278, CP-3401, and T17L in Figs. 2A and S2A and Table S1). (A) With 1 min 5

PCR extension, the fragments of wild-type and Tos17-insertion DNA were 762 (a) and 532 (b) bp, 6

respectively. (B) PCR products were used to genotype the ND2104 locus. w, wild-type (+/+); h, 7

heterozygous (+/–); T17, Tos17 homozygous (–/–). 8

9

A

B

1

2

3

4

5

1

Supplemental Figure S3. Phenotype of plants homozygous for the Tos17 insertion. (A) 2

Morphology of wild-type plants (wt +/+), Tos17-insertion heterozygotes (cap1 +/–), and 3

homozygotes (cap1 –/–). (B) Anthers of wild-type and homozygous cap1 mutant at stage 13 were 4

stained with I2-KI solution. Arrows indicate starch-filled pollen grains. Bar = 200 µm. (C) Almost 5

all pollen grains of homozygous plants were collapsed, and no starch granules were detected. Bar = 6

200 µm. (D–I) Bright-field images of anther locules from wild-type (D, E and F) and homozygous 7

cap1 (G, H and I) plants at microspore (D and G; stage 10), bicellular pollen (E and H; stage 11) 8

and tricellular pollen (F and I; stage 13) stages. Transverse-sections of anthers were stained with 9

0.1% toluidine blue. BP, bicellular pollen; Ep, epidermis; En, endothecium; MS, microspore; Ta, 10

tapetum; TP, tricellular pollen. Bar = 50 µm. (J–P) Fluorescence images of DAPI-stained 11

developing pollen. Microspores (J and M; stage 10), and bicellular (K and N; stage 11) and 12

tricellular (L and O; stage 13) pollen grains were isolated from wild-type (J–L) and homozygous 13

cap1 (M–O) plants. Arrowheads indicate stained nuclei. Arrows in N and O indicate collapsed 14

mutant pollen grains. Bars = 50 µm. The rates of normal or aberrant pollen are shown in P. Error 15

bars indicate standard deviation. Mi, microsopore (stage 10); Bi, bicellular pollen (stage 11); Tr, 16

tricellular pollen (stage 12-13); Ab, abnormal collapsed pollen with no visible nuclei. 17

18

19

1

Supplemental Figure S4. Tissue- and development-specific expression data of CAP1 and 2

closely-related genes in rice. Expression profiles of CAP1 (A, Os02g0141300) and OsARA1 genes 3

(B, Os06g0702500) were obtained from the RiceXpro Browser. Signals from anthers are shown in 4

red bars (Asterisks). 5

*

*

A

B

1

2

Supplemental Figure S5. Tissue- and development-specific expression data of CAP1-related genes 3

in Arabidopsis. Expression profiles of AtARA2 (A, At3g42850) and AtARA1 (B, At4g16130) were 4

obtained from the Arabidopsis Electronic Fluorescent Pictograph (eFP) Browser. Signals from 5

pollen grains are shown in pale blue (Asterisks). 6

7

8

A

B

*

*

Supplemental Table S1. PCR primers and oligonucleotides used in this study 1

2

Purpose Oligo name Sequence (5’-3’)

NF1037 genotyping CP-2284

CP-2798

TGAGGTGCGTACAACTCTGC

TGGTCACAAATGTTGTCCAGA

ND2104 genotyping CP-2278

CP-3401

GCCATTGTGATGGTTTCCTC

GAGTTCCCAAACAACCCTCA

NC3090 genotyping CP-5167

CP-5633

TCGAGAGGCTTGTCATGTTG

ATATGCAGCCCATCTGAACC

NG3477, NG4590 genotyping CP-6091

CP-6755

TGCTATTGCTGCTGCCTATG

ACCAACACTGCATGAACCAA

NG0590 genotyping CP-6774

CP-7403

TTGGTTCATGCAGTGTTGGT

AAACCATGCGCTCTCAAGAT

Tos17 left border, genotyping T17L ATTGTTAGGTTGCAAGTTAGTTAAGA

Tos17 probe PA0131

PA0132

TGAAGCATCGGTCTCAGCTA

GTAGGTTGGGAGGGTTGTGA

CAP1 RT-PCR CP-8328

CP-8783

TCTGGTGCAGACTTCCATTG

TGGCATCAACTTCTGCAAAC

OsUbiq1 RT-PCR 5-RUbi

3-RUbi

TGAAGACCCTGACTGGGAAG

CACGGTTCAACAACATCCAG

Cloning of Tos17 flanking

region

AP1

AP2

gwTos1

gwTos2

GTAATACGACTCACTATAGGGC

ACTATAGGGCACGCGTGGT

GAGAGCATCATCGGTTACATCTTCTCA

CTAATGTACTGTATAGTTGGCCCATGTC

3

4

5