Three CCT domain-containing genes were identified to regulate heading date by
candidate gene-based association mapping and transformation in rice
Li Zhang, Qiuping Li, Haijiao Dong, Qin He, Liwen Liang, Cong Tan, Zhongmin Han
Wen Yao, Guangwei Li, Hu Zhao, Weibo Xie, Yongzhong Xing*
Supporting materials
Table S1 Information of 107 wild rice accessions used in this study
Accession
Sources
Accession Sources
Accession
Sources
S1001
GAAS
11SW5
S1007
GAAS
11SW6
GAAS
IRGC104833
IRRI
GAAS
IRGC105568
IRRI
S1016
GAAS
11SW7
GAAS
IRGC105569
IRRI
S1020
GAAS
11SW8
GAAS
IRGC105735
IRRI
S1041
GAAS
11SW9
GAAS
IRGC105956
IRRI
S1054
GAAS
11SW10
GAAS
IRGC81886
IRRI
S1062
GAAS
11SW11
GAAS
IRGC81900
IRRI
S1064
GAAS
11SW12
GAAS
IRGC81989
IRRI
S1067
GAAS
11SW13
GAAS
IRGC93216
IRRI
S1069
GAAS
11SW14
GAAS
IRGC104599
IRRI
S1081
GAAS
11SW15
GAAS
IRGC104709
IRRI
S1084
GAAS
dongyezhangtang-7
GAAS
07YR09
HZAU
S1091
GAAS
dongyezhangtang-6
GAAS
CLR
HZAU
S1098
GAAS
dongyedongtang-1
GAAS
S2032
HZAU
S1102
GAAS
dongyeshuitaoshuixia-1
GAAS
IRGC80643
IRRI
S1107
GAAS
Linchanganjiashan-2
GAAS
IRGC80752
IRRI
S1112
GAAS
dongyeyechansi-2
GAAS
IRGC81985
IRRI
S1124
GAAS
dongyekanxialong-3
GAAS
IRGC83795
IRRI
S1133
GAAS
dongyuanlinchang-1
GAAS
IRGC103844
IRRI
S1141
GAAS
dongyedongtangshang-1
GAAS
IRGC104624
IRRI
S1143
GAAS
dongyedongtangxia-2
GAAS
IRGC104647
IRRI
S1160
GAAS
Y079
HAAS
IRGC105314
IRRI
S1212
GAAS
Y117
HAAS
IRGC105349
IRRI
S1216
GAAS
Y118
HAAS
IRGC105375
IRRI
S1192
GAAS
Y120
HAAS
IRGC105422
IRRI
S1204
GAAS
Y131
HAAS
IRGC105569
IRRI
S1109
GAAS
Y149
HAAS
IRGC105709
IRRI
S03005
GAAS
Y182
HAAS
IRGC105738
IRRI
S03013
GAAS
Y190
HAAS
IRGC105829
IRRI
S03035
GAAS
Y204
HAAS
IRGC105887
IRRI
S03038
GAAS
Y279
HAAS
IRGC105960
IRRI
S03045
GAAS
IRGC81984
IRRI
IRGC106078
IRRI
11SW1
GAAS
IRGC100916
IRRI
IRGC106424
IRRI
11SW2
GAAS
IRGC103308
IRRI
IRGC106452
IRRI
11SW3
GAAS
IRGC103423
IRRI
YSD7
JAAS
11SW4
GAAS
IRGC104501
IRRI
GAAS: Guangdong Academy of Agricultural Sciences; HAAS: Hunan Academy of Agricultural
Sciences; HZAU: Huazhong Agricultural university; IRRI: International Rice Research Institute;
JAAS: Jiangxi Academy of Agricultural Sciences.
Table S2 List of 41 CCT domain genes identified in rice
CCT-ID
OsCCT01
OsCCT02
OsCCT03
OsCCT04
OsCCT05
OsCCT06
OsCCT07
OsCCT08
OsCCT09
OsCCT10
OsCCT11
OsCCT12
OsCCT13
OsCCT14
OsCCT15
OsCCT16
OsCCT17
OsCCT18
OsCCT19
OsCCT20
OsCCT21
Chr
1
2
2
2
2
2
2
2
2
3
3
3
3
3
3
4
5
5
6
6
6
LOC
Os01g61900
Os02g01990
Os02g05470
Os02g05510＊
Os02g08150
Os02g39710
Os02g40510
Os02g49230
Os02g49880
Os03g04620
Os03g17570
Os03g22770
Os03g47970＊
Os03g50310
Os03g52450＊
Os04g42020
Os05g38990
Os05g51690
Os06g01340
Os06g15330
Os06g16370
SubC
CMF
CMF
CMF
CMF
COL
COL
PRR
COL
COL
CMF
PRR
COL
CMF
COL
CMF
COL
CMF
CMF
COL
COL
COL
CCT-ID
OsCCT22
OsCCT23
OsCCT24
OsCCT25
OsCCT26
OsCCT27
OsCCT28
OsCCT29
OsCCT30
OsCCT31
OsCCT32
OsCCT33
OsCCT34
OsCCT35
OsCCT36
OsCCT37
OsCCT38
OsCCT39
OsCCT40
OsCCT41
Chr
6
6
6
6
7
7
7
8
8
9
9
9
10
10
11
11
11
12
12
12
＊These 6 genes did not reported by Cockram et al. (2012).
LOC
Os06g19444
Os06g44450
Os06g48534＊
Os06g48610
Os07g15770
Os07g47140
Os07g49460
Os08g15050
Os08g42440
Os09g06464
Os09g33550
Os09g36220
Os10g32900
Os10g41100
Os11g01074.4
Os11g01100＊
Os11g05930
Os12g01080
Os12g01100＊
Os12g16160
SubC
COL
COL
CMF
CMF
CMF
COL
PRR
COL
COL
COL
COL
PRR
CMF
CMF
CMF
CMF
PRR
CMF
CMF
CMF
Table S3 Primers used for amplifying gene fragments and transcriptional
expression analysis
Gene
PCR
Forward primer
Reverse primer
OsCCT01
OsCCT01
OsCCT03
OsCCT03
OsCCT05
OsCCT07
OsCCT07
OsCCT09
OsCCT10
OsCCT11
OsCCT17
OsCCT18
OsCCT19
OsCCT19
OsCCT22
OsCCT22
OsCCT29
OsCCT33
OsCCT37
OsCCT38
OsCCT39
OsCCT39
OsCCT40
OsCCT41
OsCCT01
OsCCT01
Hd1
Ehd1
Hd3a
RFT1
OsCCT01
OX
RNAi
OX
RNAi
RNAi
OX
RNAi
OX
OX
RNAi
RNAi
OX
OX
RNAi
OX
RNAi
RNAi
RNAi
OX
RNAi
OX
RNAi
OX
RNAi
SC
EP
qPCR
qPCR
qPCR
qPCR
qPCR
ggtacctgaccctaccacctctcacag
ggactagtggtaccctgttgtgcaggaaatgttac
cccgggtctgaaaggcgttctgctga
aaaactagtggtaccaaggtttgttaggagcccatc
actagtggtaccaaccagatgtcctcgtcga
ggtaccctgtgctgttcgtgctgatt
actagtggtaccacttgcatctggagcgaagc
ggtaccagatcgagtgcaagtgagctgc
ggtacccgatatctcacgcccatact
ggactagtggtatttcaggtggccataatgga
actagtggtaccagatagcaggccaagggt
ggtacctccactgtctccgttcattg
ggatccgggtaatagtcgagaagggtt
aaactagtggtaccttcagcaagcagatcaagtatgc
aacgggtacctgcattcagctaatgctctttctcg
actagtggtaccggtatgcatctcgcaaggct
actagtggtaccaccgcaagttccagaagacca
actagtggtaccttgatgggcagccattctgg
ggatcctgtggacctttagattgc
actagtggtaccctgctataccctaccactacg
ggatcctgttgggctgatgtatgt
actagtggtaccgacgaataataatgtgccggaggat
cccttcctcctcactcccactg
ggactagtggtacctatgcttgccggaagacgctc
tctagaatgttccgccattcctcctctg
ggatccatcacctgttctagtcacac
tcagcaacagcatatctttctcatca
tggaaatctcgaaaaacccg
gctcactatcatcatccagcatg
tgacctagattcaaagtctaatcctt
tacctcgacggcaatgttag
ggatccccactctgacctgacctgac
cgagctcggatccgattaattacctctgctcgc
tctagacgtagtttccaggcgagtt
aaagagctcggatccgcacattgtaaacctgagac
gagctcggatccgctaatcctatgcatggtga
gtcgacctaatgagttgcaaagtgagcag
gagctcggatccccgttcagaccgactacatg
gtcgacgagtccatgaggcatacgatcg
ggatcccacttctcttgcgaatagag
cgagctcggatcttcactggttcgatcctggt
gagctcggatcccgaagttacactacggcga
ggatcctcactggcagctcgtcctct
gtcgacaaggtccagaagatgaaagaat
aaagagctcggatccgataggagtatcaatgagc
aacggtcgacttgatgcttgctaacctaacacttgg
gagctcggatcccacagcaagggttggtggat
gagctcggatccaccattagcatgacggcga
gagctcggatccggttggaagagctgtcacag
ctgcagtgttgctcacgcttgtat
gagctcggatcctactgcagttctggacaccg
aagcttaggtgaccgtctatgaaa
gagctcggatccgagggtgtaaattgaaccgtctgca
ggccgatctagatcattgagg
cgagctcggatccatcctagcagctactacgaatc
tctagacgaggcgaggttcacggacag
aagctttgtgcgctggcttatggtg
tctggaatttggcatatctatcacc
gcgctagcaaagcttcggt
ccttgctcagctatttaattgcataa
tgccggccatgtcaaattaataac
ctgatgctcctctggatgtt
OX and RNAi, primers used to amplify the gene fragmentsfor overexpression and RNA
interference,respectively.SCprimers used for protein subcellular localization,EP primers
used for gene expression patterns.
Table S4 Transformation information of 18CCT genes
CCT-ID
OsCCT01
OsCCT03
OsCCT05
OsCCT07
OsCCT09
OsCCT10
OsCCT11
OsCCT17
OsCCT18
OsCCT19
OsCCT22
OsCCT29
OsCCT33
OsCCT37
OsCCT38
OsCCT39
OsCCT40
OsCCT41
Donor
Minghui63
Nipponbare
Minghui63
Minghui 63
Minghui63
Nipponbare
Minghui63
Nipponbare
Nipponbare
Nipponbare
Minghui63
Minghui63
Nipponbare
Minghui63
Nipponbare
Minghui63
Nipponbare
Nipponbare
Receptor
Zhonghua 11
Hejiang19
Zhonghua 11
Zhonghua 11
Zhonghua 11
Zhonghua 11
Zhonghua 11
Zhonghua 11
Hejiang19
Hejiang19
Zhonghua 11
Zhonghua 11
Zhonghua 11
Zhonghua 11
Zhonghua 11
Zhonghua 11
Zhonghua 11
Zhonghua 11
Vector
1301s,ds1301
1301s,ds1301
ds1301
1301s, ds1301
1301s
1301s
ds1301
ds1301
1301s
1301s,ds1301
1301s,ds1301
ds1301
ds1301
1301s
ds1301
1301s,ds1301
1301s
ds1301
Gene fragments
DNA
CDS
DNA
DNA
DNA
DNA
DNA
DNA
DNA
DNA
CDS
DNA
CDS
DNA
CDS
DNA
DNA
DNA
Table S5 List of 59 heading date QTLs identified in rice
QTL-I
D
Qhd01
Qhd02
Qhd03
Qhd04
Qhd05
Qhd06
Qhd07
Qhd08
Qhd09
Qhd10
Qhd11
Qhd12
Qhd13
Qhd14
Qhd15
Qhd16
Qhd17
Qhd18
Qhd19
Qhd20
Qhd21
Qhd22
Qhd23
Qhd24
Qhd25
Qhd26
Qhd27
Qhd28
Qhd29
Qhd30
QTLs
Chr.
Reference
QHd1a
QHd1b
AQEA470
AQFW073
qDTH-1
AQEA472
hd1
Hd1a
dth2.1
QTL2a
AQEA490
QTL2b
AQEA506
DTH2
QHd2b
Hd9
Hd8
hd3
QHd3b
Hd6
dth4.1
AQEA499
dth4.2
AQFW133
QHd5a
QAFW190
qHD-5
QHd5b
Hd6a
Hd6b
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
3
3
3
3
3
4
4
4
4
5
5
5
5
6
6
1
3
1
3
4
1
6
3
8
5
1
5
1
11
1
12
14
16
3
17
18
1
18
3
1
3
21
1
23
23
QTL-I
D
Qhd31
Qhd32
Qhd33
Qhd34
Qhd35
Qhd36
Qhd37
Qhd38
Qhd39
Qhd40
Qhd41
Qhd42
Qhd43
Qhd44
Qhd45
Qhd46
Qhd47
Qhd48
Qhd49
Qhd50
Qhd51
Qhd52
Qhd53
Qhd54
Qhd55
Qhd56
Qhd57
Qhd58
Qhd59
QTLs
Chr.
Reference
Hd1
AQFW132
AQEA572
AQEA469
QTL7a
Ghd7
AQFW072
Hd2
Ghd7.1
Ghd8
QHd8
QHd8a
qDTH-8
QHd8b
QHd9
qHDD9-1
qDTH-9
qDTH-10
Hd-10b
qQTL-10b
AQAT006
AQEA601
QHd11
Dth11
hd11
AQEA607
dth12.1
QHd12
AQFW196
6
6
6
6
7
7
7
7
7
8
8
8
8
8
9
9
9
10
10
10
10
11
11
11
11
11
12
12
12
2
3
1
1
5
2
3
7
9
10
3
1
4
1
1
13
15
4
6
5
19
1
3
20
16
1
22
1
3
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Table S6 The genomic nucleotide diversity of CCT family
107common wild rice accessions
CCT-ID
SNP
π (10-3)
πc/πw
Θ (10-3)
sites
c
w
c
w
OsCCT01
42
2.9
2.9
0.99
1.3
2.0
OsCCT02
67
2.1
3.2
0.66
2.3
2.9
OsCCT03
42
1.3
0.9
1.44
0.9
1.2
OsCCT04
66
2.0
1.6
1.17
1.2
1.8
OsCCT05
82
2.9
3.6
0.81
2.0
3.6
OsCCT06
76
3.4
2.6
1.31
2.2
3.3
OsCCT07
123
2.7
2.9
0.93
2.7
3.2
OsCCT08
137
2.6
2.7
0.96
1.5
2.3
OsCCT09
87
2.1
2.0
1.10
1.2
2.4
OsCCT10
77
1.1
1.6
0.69
0.9
1.4
OsCCT11
211
1.6
2.5
0.64
1.3
2.4
OsCCT12
150
2.0
4.9
0.41
3.0
5.4
OsCCT13
89
2.5
2.4
1.04
1.5
2.2
OsCCT14
95
2.5
3.8
0.66
2.0
3.2
OsCCT15
77
1.8
1.4
1.29
1.0
1.5
OsCCT16
75
2.2
4.5
0.49
1.3
3.6
OsCCT17
34
1.5
1.7
0.84
0.5
1.2
OsCCT18
94
0.1
2.6
0.04
1.1
2.3
OsCCT19
61
3.5
2.8
1.25
2.0
3.2
OsCCT20
82
6.0
4.1
1.46
2.1
3.3
OsCCT21
105
2.3
2.3
0.99
1.2
3.8
OsCCT22
96
2.6
2.3
1.09
1.4
1.9
OsCCT23
49
2.6
2.5
1.04
1.5
2.0
OsCCT24
92
3.7
3.4
1.09
1.6
2.2
OsCCT25
146
3.6
2.5
1.44
2.2
3.7
OsCCT26
165
6.0
5.5
1.09
3.2
5.4
OsCCT27
129
2.2
4.0
0.55
1.5
4.1
OsCCT28
199
1.9
1.8
1.06
1.1
1.7
OsCCT29
150
7.8
8.4
0.93
5.7
7.1
OsCCT30
92
1.9
1.8
1.06
1.1
1.7
OsCCT31
421
3.0
3.2
0.94
1.9
3.1
OsCCT32
80
2.5
1.5
1.67
1.7
2.0
OsCCT33
123
1.9
2.4
0.79
1.5
2.5
OsCCT34
55
2.5
1.9
1.32
1.3
1.9
OsCCT35
72
2.4
2.7
0.89
1.2
2.5
OsCCT36
10
0.3
0.2
1.50
0.2
0.2
OsCCT37
20
2.6
2.1
1.24
1.6
1.8
OsCCT38
121
2.3
2.4
0.96
1.5
2.4
OsCCT40
22
2.7
2.3
1.17
1.8
2.2
OsCCT41
88
4.8
3.1
1.55
3.3
3.8
genes in 529 cultivars and
Tajima’s D
c
w
2.89*
1.20
-0.29
0.33
1.28
-0.85
1.74
-0.38
1.22
0.21
1.42
-0.69
-0.03
-0.24
2.03
0.52
1.94
-0.50
1.29
0.47
0.79
0.05
-0.95
-0.28
1.87
0.32
0.56
0.59
2.11
-0.28
1.63
0.72
3.89** 1.05
-2.31* 0.47
1.91
-0.39
4.97** 0.68
2.42
-1.25
2.14
0.68
1.96
0.79
3.66*
1.62
1.90
-1.01
2.52
0.01
1.35
-1.01
2.02
0.23
1.05
0.57
2.16
0.06
1.66
0.12
1.24
-0.80
0.80
-0.12
2.42
-0.03
2.36
0.20
2.03
0.56
1.44
0.40
1.46
0.03
1.10
0.18
1.28
-0.59
π, average number of nucleotide differences per site between two sequences; θ, Watterson
estimator; Tajima’s D, test for neutral selection； *and ** Significant at P<0.01 and
P<0.001 respectively.
πc and πw indicate π in cultivars and π in wild rice respectively.
Table S7 The nucleotide diversity of noncoding regions around OsCCT18 in
cultivars and wild rice
PositionS
(bp)
29564885
29623975
29633070
29644549
29654549
29667564
29680439
29700011
29710059
Regions
IG
IT
IT
IG
OsCCT18
IG
IT
IG
IG
N
529
529
529
529
529
529
529
529
529
Cultivars
L
S
2055 30
313
4
409
3
948
27
5726 45
805
13
906
5
1995 38
2013 18
H
68
7
5
15
27
11
6
45
27
N
107
107
107
107
107
107
107
107
107
Wild rice
L
S
2055 39
313
5
409
4
948
47
5726 71
805
24
906
8
1995 58
2013 22
πc/πw
H
103
11
6
103
166
35
10
133
95
0.83
0.33
0.14
0.08
0.04
0.18
0.07
0.11
0.10
N: sample size; L: the length in bp from the first SNP to the last SNP; S: number of
segregating sites; H: number of haplotypes; π: average proportion of pairwise differences
per base pair. IG: intergenic region; IT: intron; πc and πw indicate π in cultivars and π in
wild rice.
Table S8 Phenotypes of OsCCT1 transgenic plants in the T2 generation under nature long
day conditions in 2012 summer (Wu Han)
Genotype
Wild type
Plant
height
(cm)
Spikelets
per panicle
100.7±3.8 239.3±24.8
Seed
setting
rate
Length of internodes (cm)
1st
2nd
3rd
87.0±8.3
37.9±1.1
14.3±0
14.9±1.9
OXOsCCT1+
69.3± 4.5
140.7±10.2
20.1±
25.0±0.9
.7 ±
9.5
6.2 ± 0.8
+
＋)
OsCCT1RNAi
98.4±1.6
220.3±20.3
6.7
90.1±7.5
36.5±1.7
0.5
13.2±1
12.1±1.8
Data are present in means ± s.d. (N=15). The internodes indicate 1st.3
, 2nd and 3rd upmost
stem internodes in the main culms.
Figure S1 Population structure for cultivars in the study. Neighbour-joining tree of cultiva
accessions, which was calculated from ～200000 SNPs randomly selected from the
whole genome. Black, Ind; red, IndI; green, IndII ; yellow, TrJ; blue, TeJ; grey, Jap.
Figure S2 Expression levels of 16 OsCCT genes in wild type (WT), negative transgenic
plant (NP) and positive transgenic plant (PP) plants. Relative expression level of each
gene compared with that of rice UBQ. Error bars indicate standard deviations, based on 3
biological replicates
OsCCT-ZH11-OX represented Zhonghua 11 plants overexpressed OsCCT gene.
OsCCT-ZH11-RNAi represented Zhonghua 11 plants suppressed OsCCT gene by RNAi.
Figure S3 Expression levels of OsCCT01, 11 and 19 in wild type (WT), negative
transgenic plant (NP) and positive transgenic plant (PP) plants.
Relative expression level of each gene was compared with that of rice UBQ. Error bars
indicate standard deviations, based on 3 biological replicates. HD means heading date
scored as days to heading from sowing.
OsCCT01-ZH11-OX,
the
genotype
Zhonghua
11
overexpressed
OsCCT01.
OsCCT11-ZH11-RNAi, the genotype Zhonghua 11 suppressed OsCCT11 by RNAi.
OsCCT19-HJ19-OX, the genotype Hejiang 19 overexpressed OsCCT19.
Figure S4 Diurnal expression patterns of OsCCT01 in Zhonghua 11
Diurnal expression patterns of the OsCCT01 in Zhonghua 11 as indicated by quantitative
RT-PCR results. In all panels the mean of each point is based on the average of two
biological repeats calculated using the relative quantification method. The black bars
indicate the dark period, and the white bars indicate the light period. The blue line
indicates the long-day condition. The red line indicates the short-day condition. The
numbers below the bars indicate hours of the day. Error bars indicate standard deviations.
Figure S5 Subcellular localization of OsCCT01
Rice protoplasts were co-transformed with the fusion constructs 35s::GHD7:CFP (a) and
35s::OsCCT01:YFP (b); (c) bright field image and (d) merged image. Bar = 12 µm
Figure S6 Diurnal expression patterns of OsGI, RID1 and Ehd3 in OsCCT01-positive
transgenic plants and wild-type Zhonghua 11 plants grown under long day-length
conditions and short day-length conditions
X axis indicated the time points for sampling. Y axis indicated the relative expression
levels of investigated genes to UBQ.
Figure S7 Diurnal expression patterns of Ehd4, OsMADS50, OsMADS56, Ghd7,
Ghd8, Ghd7.1 and DTH2 in OsCCT01-positive transgenic plants and wild-type
Zhonghua 11 plants grown under long day-length conditions, Ehd4, OsMADS51
under short day-length conditions
X axis indicated the time points for sampling. Y axis indicated the relative expression
levels of investigated genes to UBQ.