SUPPLEMENTARY TABLES
Table S1 T-DNA alleles in the LORELEI gene, but not those in flanking
genomic sequences, segregate reduced seed-setting plants
Seed set phenotype in
Mutant allele
self-fertilized progeny
designations
T-DNA line
lre allele
of lre mutants
in Figure 1
Full
Reduced
(b)
SAIL_8_D07*
lre-4
7
14
(c)
SAIL_109_C01*
lre-6
4
12
(d)
SAIL_245_B10*
lre-7
14
25
(e)
SAIL_251_E06
lre-8
15
10
(f)
SAIL_109_E01
lre-9
3
14
(g)
SALK_011553
65
0
(h)
SAIL_1159_G11
56
0
(i)
SAIL_350_D03
30
0
(j)
SALK_042505C
32
0
*
A reduced seed-setting plant from these lines that was confirmed to
contain the T-DNA by PCR and shown to segregate 2 GUS-: 2 GUS+ in
pollen tetrads (Johnson et al., 2004) was self-fertilized and the progeny
were used for (i) linkage and mutant allele transmission experiments
described in Tables S5 and S6 and (ii) phenotype characterization
experiments described in this study.
Table S2 The lre-5 mutation is transmitted at a reduced frequency through female gametophytes
Female
parent
Male
parent
Progeny phenotypea
Transmission
efficiencyb (%)
F
R
VR
(LRE/LRE) (LRE/lre-5) (lre-5/lre-5)
NA
LRE/lre-5
LRE/lre-5
Self c
89
95
16
LRE/lre-5
LRE/LRE
Manuald
90
10
NA
9.0
LRE/LRE
LRE/lre-5
Manuale
147
134
NA
91.1
a
F, full seed-setting plants = >99% normal seeds (>60 seeds/silique); R, reduced seed-setting plants =
~60% normal seeds (~40 seeds/silique); VR, very reduced seed-setting plants = <25% normal seeds (<20
seeds/silique).
b
Transmission efficiency = (LRE/lre-5 plants)/(LRE/LRE plants) x 100.
c
Progeny phenotype segregation significantly deviated from expected ratio of 1:2:1; 2, P < 0.01.
d
Progeny phenotype segregation significantly deviated from expected ratio of 1:1; 2, P < 0.01.
e
Progeny phenotype segregation did not deviate significantly from expected ratio of 1:1; 2, P >0.01.
NA, not applicable.
Pollination
Table S3 The lre-5 mutation is transmitted at a reduced frequency through female gametophytes and kanamycin resistance is linked to
the reduced fertility phenotype in lre-5 mutants
Kanamycin-sensitivity
Segregation of seed set
KanR
phenotype
phenotype in KanR plantsb
Female
Male
Transmission
plants
Pollination
Sensitive
Resistant
parent
parent
efficiencya (%) moved
F
R
VR
(LRE/LRE) (LRE/lre-5 or
to soil (LRE/LRE) (LRE/lre-5) (lre-5/lre-5)
lre-5/lre-5)
LRE/lre-5 LRE/lre-5 Selfc
124
177
NA
176
0
153
23
LRE/lre-5 LRE/LRE
Manuald
144
11
7.63
7
0
7
0
e
LRE/LRE
LRE/lre-5 Manual
214
173
80.84
139
0
139
0
aTransmission efficiency = (kanamycin-resistant plants)/(kanamycin-sensitive plants) x 100.
bSeed-setting phenotype was scored as described in Table S2 (F, full seed-setting plants; R, reduced seed-setting plants; VR, very reduced
seed-setting plants).
cSegregation of kanamycin resistance in progeny significantly deviated from expected ratio of 1:3 (sensitive : resistant; 2, P <0.01).
dSegregation of kanamycin resistance in progeny significantly deviated from expected ratio of 1:1 (sensitive : resistant; 2, P <0.01).
eSegregation of kanamycin resistance in progeny did not deviate significantly from expected ratio of 1:1 (sensitive : resistant; 2, P
>0.01). KanR, kanamycin resistant.
NA, not applicable.
Table S4 lre-5 mutation does not affect cell-specification of the female gametophytic cells
Percent Percent
FG cell
Number of
Number of
ovules
ovules
Plant genotype
marked with
ovules with
ovules
Pa
with
without
GFP
GFP
without GFP
GFP
GFP
DD45:GFP/+, LRE/LRE
Egg cell
164
58
73.9
26.1
0.9>P>0.5
DD45:GFP/+, LRE/lre-5
Egg cell
232
77
75.1
24.9
DD3:GFP/+, LRE/LRE
Synergid cell
67
136
33.0
67.0
0.5>P>0.1
DD3:GFP/+, LRE/lre-5
Synergid cell
87
189
31.5
68.5
DD11:GFP/+, LRE/LRE
Synergid cell
102
30
77.3
22.7
0.9>P>0.5
DD11:GFP/+, LRE/lre-5
Synergid cell
297
91
76.6
23.5
DD19:GFP/+, LRE/LRE
Central cell
92
65
58.6
41.4
0.9>P>0.5
DD19:GFP/+, LRE/lre-5
Central cell
211
156
57.5
42.5
DD22:GFP/+, LRE/LRE
Central cell
81
94
46.3
53.7
0.9>P>0.5
DD22:GFP/+, LRE/lre-5
Central cell
120
147
44.9
55.1
DD6:GFP/+, LRE/LRE
Antipodal cell
60
69
46.5
53.5
0.9
DD6:GFP/+, LRE/lre-5
Antipodal cell
116
136
46.0
54.0
a
Chi-square test was performed to evaluate if the ratio of ovules with GFP : ovules without GFP in marker lines
with lre-5 mutation deviated significantly from ratio of ovules with GFP : ovules without GFP in corresponding
control marker lines that did not carry the lre-5 mutation.
FG, female gametophyte.
Table S5 Basta resistance, pollen tetrad GUS expression and reduced seed set phenotypes are
linked in lre-4, lre-6 and lre-7 mutants
Basta-sensitivity
Number of
Segregation in BastaR progenyb
phenotypea
Genotype
BastaR
of parent
plants
2 GUS-:2 GUS+
0 GUS-:4 GUS+
plant
(LRE/lre)
(lre/lre)
Sensitive Resistant moved to
soil
F
R
VR
F
R
VR
118
28
LRE/lre-4
100
202
146
0
0
0
0
127
23
LRE/lre-6
203
198
150
0
0
0
0
122
25
LRE/lre-7
249
396
147
0
0
0
0
a
Progeny phenotype segregation significantly deviated from expected ratio of 1:3 (sensitive :
resistant); 2, P <0.01.
b
Segregation of GUS expression in pollen tetrads [GUS-, pollen with no GUS staining; GUS+,
pollen with GUS stain; (Johnson et al., 2004)] and silique seed-setting phenotypes (as described in
Table S2; F, full seed-setting plants; R, reduced seed-setting plants; VR, very reduced seed-setting
plants) were used to assign progeny genotypes (within parentheses).
BastaR, Basta resistant.
Table S6 Distorted segregation of pollen tetrad GUS expression and reduced seed set
phenotypes in progeny of self-fertilized lre-4, lre-6 and lre-7 mutants
Segregation of phenotypesa, b
4 GUS-:0 GUS+
(LRE/LRE)
Plant
genotype
F
R
VR
2 GUS-:2 GUS+
(LRE/lre)
F
R
VR
0 GUS-:4 GUS+
(lre/lre)
F
R
VR
LRE/lre-4
57
0
0
0
67
0
0
0
23
LRE/lre-6
59
0
0
0
71
0
0
0
22
LRE/lre-7
57
0
0
0
77
0
0
0
16
a
Segregation of GUS expression in pollen tetrads [GUS , pollen with no GUS staining;
GUS+, pollen with GUS stain; (Johnson et al., 2004)] and silique seed-setting
phenotypes (as described in Table S2; F, full seed-setting plants; R, reduced seedsetting plants; VR, very reduced seed-setting plants) were used to assign progeny
genotypes (within parentheses).
b
Segregation of both phenotypes significantly deviated from expected ratio of
1:2:1;.2, P <0.01.
Table S7 Primers used in this study
Experiment
Primer Name
Primer
numbers
Sequence (5'-3')
Reference
Product Size
in Figures
1 and 8
SALK-RB-10256FPrimary
SALK-RB-10281FSecondary
SALK-RB-10330FTertiary
TAIL-PCR of lre-5
locus
To amplify RB-genomic
DNA junction in lre-5
To amplify LB-genomic
DNA junction in lre-5
To amplify genomic
region spanning T-DNA
insertion site in lre-5
To amplify LB-genomic
DNA junction in lre-4
To amplify LB-genomic
DNA junction in lre-4
To amplify LB-genomic
DNA junction in lre-6
To amplify LB-genomic
DNA junction in lre-6
To amplify LB-genomic
DNA junction in lre-7
To amplify LB-genomic
DNA junction in lre-7
To amplify LB-genomic
DNA junction in llg1-1
To amplify LB-genomic
DNA junction in llg1-1
To amplify LB-genomic
DNA junction in llg1-2
To amplify RB-genomic
DNA junction in llg1-2
SSLP marker
CAPS marker
(bp)
1
TGAGTGGCTCCTTCAACGTTG
This study
2
TCTGTCAGTTCCAAACGTAAAAC
This study
3
TCATAACGTGACTCCCTTAATTC
This study
AD1
NGTCGASWGANAWGAA
AD2
TGWGNAGSANCASAGA
AD3
AGWGNAGWANCAWAGG
AD6
WGTGNAGWANCANAGA
AD7
WGGWANCWGAWANGCA
AD8
WCGWWGAWCANGNCGA
AD9
WGCNAGTNAGWANAAG
(Liu et al.,
1995)
(Liu et al.,
1995)
(Liu et al.,
1995)
(Liu et al.,
1995)
(Liu et al.,
1995)
(Liu et al.,
1995)
(Liu et al.,
1995)
(Liu et al.,
1995)
~2500 (Secondary);
~2400 (tertiary)
~1100 (Secondary);
~1000 (tertiary)
~1600 (Secondary);
~1500 (tertiary)
~1100 (Secondary);
~1000 (tertiary)
~1500 (Secondary);
~1400 (tertiary)
~1200 (Secondary);
~1100 (tertiary)
~2100 (Secondary);
~2000 (tertiary)
~200 (Secondary);
~100 (tertiary)
This study
~400
This study
~350
This study
500 (WT);
~4500 (lre-5)
This study
~450
This study
~1000
This study
~350
This study
~500
This study
~800
This study
~800
This study
~250
This study
~200
This study
~520
This study
~1200
This study
~300
(Salathia et al.,
2007)
Col:317, Ler:284
This study
Enzyme: FokI
Col:473+282,
Ler:755
AD10
6
AWGCANGNCWGANATA
SALK-RB-10256F
LORELEI-R2
SALK-LB-6363R
LORELEI-upF1
1
5
4
6
TGAGTGGCTCCTTCAACGTTG
CGGATTAACAAATTAAAAGGATG
CGGGCTATTCTTTTGATTTATAAG
AAGCCAGTTTTAGAGTACGAAGA
LORELEI-R2
5
CGGATTAACAAATTAAAAGGATG
LORELEI-upF1
6
AAGCCAGTTTTAGAGTACGAAGA
SAIL-LB-423R
LORELEI-R1
SAIL-LB-423R
LORELEI-upF1
SAIL-LB-423R
LORELEI-R2
SAIL-LB-423R
LORELEI-upF1
SAIL-LB-423R
LORELEI-R1
SAIL-LB-423R
LORELEI-upF1
SAIL-LB-423R
5g56170-1F
SAIL-LB-423R
5g56170-59F
SAIL-LB-423R
5g56170-509R
SALK-LB-6363R
5g56170-59F
SALK-RB-10256F
5g56170-1027R
4g25400-786F
4g25400-downR
4g27270-242F
7
8
7
6
7
5
7
6
7
8
7
6
7
11
7
12
7
13
4
12
1
15
GCTTCCTATTATATCTTCCCAAATTACCAATACA
TCAAGTCAACACTAACAAAGCAAAAACAGCGG
GCTTCCTATTATATCTTCCCAAATTACCAATACA
AAGCCAGTTTTAGAGTACGAAGA
GCTTCCTATTATATCTTCCCAAATTACCAATACA
CGGATTAACAAATTAAAAGGATG
GCTTCCTATTATATCTTCCCAAATTACCAATACA
AAGCCAGTTTTAGAGTACGAAGA
GCTTCCTATTATATCTTCCCAAATTACCAATACA
TCAAGTCAACACTAACAAAGCAAAAACAGCGG
GCTTCCTATTATATCTTCCCAAATTACCAATACA
AAGCCAGTTTTAGAGTACGAAGA
GCTTCCTATTATATCTTCCCAAATTACCAATACA
ATGGAGCTCCTCTCTAGAGCTC
GCTTCCTATTATATCTTCCCAAATTACCAATACA
TCTCTTCTTCAAGTTTCATTTCAG
GCTTCCTATTATATCTTCCCAAATTACCAATACA
TCAAGAGGATCATCATTAAGCCAC
CGGGCTATTCTTTTGATTTATAAG
TCTCTTCTTCAAGTTTCATTTCAG
TGAGTGGCTCCTTCAACGTTG
TTAACAAAAACCAAAAGAGCCG
GATCTTGTCGGCTTACAAACG
CATCGTTTTGGTTTGAGAAGTT
ATTAGAATCATGGATTTGTGTTG
CTGAACACAAAGGAAATAACTTG
4g27270-996R
Table S7 Continued…
Experiment
Primer Name
Primer
numbers
in Figures
1 and 8
Sequence (5'-3')
Reference
Product Size
(bp)
RT-PCR and qRT-PCR
RT-PCR and qRT-PCR
(Figures 7 and S2)
RT-PCR (Figure 8)
RT-PCR and qRT-PCR
RT-PCR and qRT-PCR
RT-PCR and qRT-PCR
(Figures 7c,d and S2)
RT-PCR (Figure 7c)
qRT- PCR (Figure 7d
and S2)
RT-PCR (Figures 7a and
S2a)
qRT-PCR (Figure S2b)
RT-PCR (Figures 7a,c,
and S2a)
qRT-PCR (Figures 7d
and S2b)
LORELEI-F1
LORELEI-R1
LLG1-F1
LLG1-R1
LLG1-F2
LLG1-R2
LLG2-F
LLG2-R
LLG3-F
LLG3-R
FERONIA-F
FERONIA-R
AMC-F1
AMC-R1
AMC-F2
AMC-R2
MYB98-F1
MYB98-R1
MYB98-F2
MYB98-R2
ACTIN2-F1
ACTIN2-R1
ACTIN2-F2
ACTIN2-R2
9
8
10
14
11
15
ATGGAGCTGATATTATTATTCTTCTTTCTGATGGC
TCAAGTCAACACTAACAAAGCAAAAACAGCGG
AAAGAACCATGGAGCTCCTCTC
TGAGCTGGTCAGTGTAAGGACA
ATGGAGCTCCTCTCTAGAGCTC
TTAACAAAAACCAAAAGAGCCG
TCCTCTCTGGTTTCTCCCTTTC
CAAGACCTTCTTTGCCCTCTTT
ATCCTCCTTTTGTCTGGATTCG
GAAGAGGTGAAACAAGATGGAAA
TGATTTTCTCTCTCTCTCCCCC
AAAGGAGATCGGAAAACTCTCG
AGGTGGCAGTCCTCCTAAACCT
TTAGTTGCCCCATACATTGTCC
TGGTTATGGTATGGGTATGGGA
AGAAATTCACAGCACCTTGCAT
CTCCTTTCCAAAACAATGGAGAATTTCGTC
GTCCTCTTCTTCACTCCATGTTTCTTTCTTA
AAGACAGGGTACTGATTCAACTCG
AGCGATATGCGACCATTTACGCAA
CCTATTGAGCATGGTGTTGTTAGCAAC
TGTGAGACACACCATCACCAGA
TCCCTCAGCACATTCCAGCAGAT
AACGATTCCTGGACCTGCCTCATC
This study
This study
This study
This study
This study
This study
This study
This study
(Kasahara et al.,
2005)
(Kasahara et al.,
2005)
This study
This study
gDNA:985,
cDNA:497
gDNA:809,
cDNA:279
gDNA:1027,
cDNA:497
gDNA:541,
cDNA:321
gDNA:661,
cDNA:441
gDNA:600,
cDNA:425
gDNA:1181,
cDNA:898
gDNA:270,
cDNA:164
gDNA:1052,
cDNA:846
gDNA:62,
cDNA:62
gDNA:355,
cDNA:277
gDNA:155,
cDNA:65