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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
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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
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5