Supplementary Materials
DDM1 (Decrease in DNA Methylation) genes in rice (Oryza sativa)
Hiromi Higo, Muhammad Tahir, Kazuya Takashima, Asuka Miura, Koichi Watanabe, Akemi
Tagiri, Masashi Ugaki, Ryuji Ishikawa, Mitsugu Eiguchi, Nori Kurata, Takuji Sasaki, Eric
Richards, Makoto Takano, Naoki Kishimoto, Tetsuji Kakutani, and Yoshiki Habu
Correspondence to:
Makoto Takano, Yoshiki Habu
E-mail: [email protected], [email protected]
Phone: +81-29-838-7442, FAX: +81-29-838-7073
Tetsuji Kakutani
E-mail: [email protected]
Phone: +81-55-981-6801, FAX: +81-55-981-6804
1
Supplementary Table 1 Primers used in this study
target
primer
sequence
method
reference
OsDDM1a
OsDDM1R2
GCTGGGTAGACAATTGTATCATGACA
RT
this study
OsDDM1F2
TTCATTCGCTCGCTGTTGCCGTCT
PCR
this study
OsDDM1R1
CACACTACTACATCACCTCCTACTC
PCR
this study
OsDDM1aF2
AATGGGGATGTCTTGGATACCACC
probe for northern
this study
OsDDM1aR2
TGAATAGAGTTGTGTCTGTGATAGT
probe for northern
this study
OsDDM1R5
GCAGGTAGACAAGGATATTATGTTCT
RT
this study
OsDDM1F4
CATCGCTCGCTGCGCACCTCAG
PCR
this study
OsDDM1R4
ACAGATTAGATGCACACTACCTTATC
PCR
this study
ddmT7-1F1
ATACATATGTGTGGCCTTCCATGCA
RT
this study
ddmT7-1F2
TGACTTCACTTCCCATCTGACAACT
PCR
this study
ddmT7-1R2
GACACATTGGATAGTGCTATGGCCT
PCR
this study
ddmT-12-1F1
CAACTTTCAATTATACGCTTTCGCTG
RT
this study
ddmT-12-1F2
TCAAACAGAACCACTATAGCCCGTC
PCR
this study
ddmT-12-1R2
TTCAGTGATTGCACGGCCATTTCC
PCR
this study
RTN12-1PF1
CAGGCAACAGCCGTGACAGGTTC
probe for Southern
this study
RTN12-1PR1
TTGCACGGCCATTTCCAAAATTGGC
probe for Southern
this study
ddmT11-2F1
ACCCTTATCAACAGAACAGCCTCTC
RT
this study
ddmT11-2F2
TCCTTCTAGCTGTTGCCAGCTTACA
PCR
this study
ddmT11-2R2
GAGATGACGGCAACACGGTGGAT
PCR
this study
RTN11-2PF2
ATCCTCCCTTCCTTGAGAGCCGG
probe for Southern
this study
RTN11-2PR1
TAAGCTATCTTTCAAGTATTTTGGTCCT
probe for Southern
this study
ddmT6-1F1
TGTCAGGATCCAACGATTTTGGTTC
RT
this study
ddmT6-1F2
GCTAGTAGCTAACTAATGGGAATCC
PCR
this study
ddmT6-1R2
GAATTGCCGTACTGTGGACGTCAT
PCR
this study
nDart1-2F1
ATGGTGCCGGCATTTTCGGAATGG
primary PCR
reference 1
nDart1-2F2
CAGGTTCATCCCCAATCCTGCAA
nested PCR
reference 1
nDart1-2R1
GCGGATTGATTTCTGGCGCAGAGA
primary PCR
reference 1
nDart1-2R2
GTTGCGATTGGGATGAGACTGTGA
nested PCR
reference 1
nDart1-3(6)F1
CGAGTAGCAAGAGCACTGCTTGA
primary PCR
reference 1
nDart1-3(6)F2
TTGTCAGGGGAGACAGAGTCGAA
nested PCR
reference 1
nDart1-3(6)R1
TGGATTTCGGTACACCGGTGAGA
primary PCR
reference 1
nDart1-3(6)R2
TTTCCACCCTCCAAACCGGAAAG
nested PCR
reference 1
DaiZ9F1
GTTTGGAGGGCTTTTGACA
primary PCR
reference 2
DaiZ9F2
ATCTCTCTAGCTGCCAGAAGCTCC
nested PCR
this study
DaiZ9R1
CCACGAACCCCACTATACG
primary PCR
reference 2
DaiZ9R2
AGACCGTCCAGACAAGCTGCGA
nested PCR
this study
RILN39-F1
GGGATTACCTCCTAGAACTCATGCG
probe for Southern
this study
RILN39-R1
CCTTGAAGCCGGGTGAGGTTACCTT
probe for Southern
this study
OsDDM1b
Os07g0431000
Os12g0428300
Os11g0677100
Os06g0493000
nDart1-2
nDart1-3(6)
DaiZ9
RILN39
2
Supplementary Table 1 Primers used in this study (continued)
target
primer
sequence
method
reference
ACTIN
OsAc2
GTACCCGCATCAGGCATCTGATTAA
RT and PCR
this study
OsAc1
TTCTTCGGACCCAAGAATGCTAAGC
PCR
this study
References
1. Tsugane et al. (2006) Plant J 45:46-57
2. Huang et al. (2009) Genomics 93:274-281
3
Supplementary Table 2 Genes up-regulated in the hypomethylated line
Gene
Description
Os01g0356900
Extensin-like protein.
Os01g0382000
Pathogenesis-related protein 1 precursor (PR-1).
Os01g0549300
Homeodomain-like containing protein.
Os01g0678000
Conserved hypothetical protein.
Os01g0692400
Conserved hypothetical protein.
Os01g0829300
(No Hit)
Os02g0129000
Conserved hypothetical protein.
Os02g0161100
Actin-binding FH2 domain containing protein.
Os02g0255400
RNA-directed DNA polymerase (Reverse transcriptase) domain containing protein.
Os02g0300000
Conserved hypothetical protein.
Os02g0569900
Cytochrome P450 family protein.
Os02g0772100
Conserved hypothetical protein.
Os02g0789700
Conserved hypothetical protein.
Os03g0115800
Conserved hypothetical protein.
Os03g0141100
Conserved hypothetical protein.
Os03g0233700
(No Hit)
Os03g0377400
Hypothetical protein.
Os03g0411900
Retrotransposon gag protein family protein.
Os03g0429900
Nucleic acid-binding OB-fold domain containing protein.
Os03g0569000
Hypothetical protein.
Os03g0629800
Conserved hypothetical protein.
Os03g0638400
Conserved hypothetical protein.
Os03g0714800
En/Spm-like transposon proteins family protein.
Os03g0790100
(No Hit)
Os04g0115200
Conserved hypothetical protein.
Os04g0178300
Copalyl diphosphate synthetase (Fragment).
Os04g0179200
Stem secoisolariciresinol dehydrogenase (Fragment).
Os04g0223600
Non-protein coding transcript, unclassifiable transcript.
Os04g0288100
Adenosine diphosphate glucose pyrophosphatase precursor.
Os04g0326000
ARM repeat fold domain containing protein.
Os04g0397800
Non-protein coding transcript, unclassifiable transcript.
Os04g0510000
Hypothetical protein.
Os05g0124700
Conserved hypothetical protein.
Os05g0232900
Conserved hypothetical protein.
Os05g0263300
Non-protein coding transcript, unclassifiable transcript.
Os05g0296200
Conserved hypothetical protein.
Os05g0374700
Retrotransposon gag protein family protein.
Os05g0417400
(No Hit)
Os05g0438300
(No Hit)
Os06g0265100
Hypothetical protein.
Os06g0271400
Hypothetical protein.
Os06g0493000
Retrotransposon gag protein family protein.
Os07g0115200
Conserved hypothetical protein.
Os07g0153000
ZIM domain containing protein.
4
Supplementary Table 2 Genes up-regulated in the hypomethylated line (continued)
Gene
Description
Os07g0195900
Polyprotein.
Os07g0220200
(No Hit)
Os07g0429600
Conserved hypothetical protein.
Os07g0429700
Conserved hypothetical protein.
Os07g0431000
En/Spm-like transposon proteins family protein.
Os07g0432300
Conserved hypothetical protein.
Os07g0442800
Conserved hypothetical protein.
Os07g0511400
Hypothetical protein.
Os07g0648500
(No Hit)
Os07g0674900
(No Hit)
Os07g0677500
Peroxidase POC1.
Os07g0694200
Conserved hypothetical protein.
Os08g0140700
Conserved hypothetical protein.
Os08g0172300
HAT dimerisation domain containing protein.
Os08g0178900
Homeodomain-like containing protein.
Os08g0240200
Hypothetical protein.
Os08g0298800
Hypothetical protein.
Os08g0344700
Retrotransposon gag protein family protein.
Os08g0367300
Conserved hypothetical protein.
Os09g0396900
Protein of unknown function DUF125 family protein.
Os09g0491900
Integrase, catalytic region domain containing protein.
Os10g0118700
(No Hit)
Os10g0343500
(No Hit)
Os10g0360700
Conserved hypothetical protein.
Os10g0371600
Conserved hypothetical protein.
Os10g0409400
BURP domain containing protein.
Os11g0224300
Integrase, catalytic region domain containing protein.
Os11g0471200
Conserved hypothetical protein.
Os11g0514400
BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 precursor (EC 2.7.1.37)
(BRI1-associated receptor kinase 1) (Somatic embryogenesis receptor-like kinase 3).
Os11g0519100
Conserved hypothetical protein.
Os11g0595900
Hypothetical protein.
Os11g0618200
(No Hit)
Os11g0636200
(No Hit)
Os11g0677100
Retrotransposon gag protein family protein.
Os12g0236800
(No Hit)
Os12g0275100
Hypothetical protein.
Os12g0275200
Conserved hypothetical protein.
Os12g0415400
Histone H3.
Os12g0415800
Histone H3.2 (Fragment).
Os12g0428300
Retrotransposon gag protein family protein.
Os12g0539800
Conserved hypothetical protein.
5
Supplementary Table 3. Numbers of transposon-related loci on the 44k microarray and activated loci in the asDDM1 line.
number of probes on the 44k
number of loci up-regulated in
microarray
the asDDM1 line*
retrotransposon-like
43
6
similarity to reverse transcriptase
60
1
En/spm-like
26
2
similarity to integrase
31
2
polyprotein-like
14
1
Mutator-like
10
0
type of transposon-related loci
*
≥10 fold increase in the asDDM1 line
6
Supplementary Figure legends
Supplementary Fig. 1 Status of cytosine methylation in transgenic rice lines transformed
with T-DNA carrying an antisense DDM1a EST or a control vector
Methylation-sensitive Southern hybridization of genomic DNA extracted from independent
rice plants transformed with the antisense OsDDM1a T-DNA (asDDM1) or control vector.
Lanes 1-8, independent transgenic rice plants transformed with the antisense OsDDM1a
EST (the primary generation); 9-11, control plants transformed with an empty vector
(pBI101); 12, the fifth generation of line 17; 13, wild type Nipponbare.
Supplementary Fig. 2 Characterization of line 17
Accumulation of antisense RNA of the EST of OsDDM1a (asDDM1; a) and mRNA of
endogenous OsDDM1 genes (OsDDM1; b) were examined by RT-PCR and RNA blot,
respectively, with two sibling progenies of line 17 (17A and 17C). The probe (444 bp) used in
the RNA blot has a sequence identical to OsDDM1a and 91% similarity to OsDDM1b, and
detects transcripts of both genes. Primers used are listed in Supplementary Table 1.
Supplementary Fig. 3 Analysis for detecting excision of endogenous transposons in the
antisense OsDDM1 line
PCR analysis for detection of excision of Daiz9 (a), and nDart1-2 and nDart1-3(6) (b). Wild
type Nipponbare (N) and three progenies of line 17 were examined. Expected sizes of the
excision products are indicated with arrowheads. Primers used are listed in Supplementary
Table 1.
7
Supplementary Fig.4. Southern analysis of transposon-like loci in the antisense
OsDDM1 line.
Three micrograms of genomic DNA were digested with NdeI and probed with genomic
fragments of Os11g0677100 (a) and Os12g0428300 (b), respectively (Fig.4b). Nipponbare
(N) and three asDDM1 plants at the 10th generation were used in the analysis. Primers
used are listed in Supplementary Table 1.
8