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: ,

Phone: +81-29-838-7442, FAX: +81-29-838-7073

Tetsuji Kakutani

E-mail:

Phone: +81-55-981-6801, FAX: +81-55-981-6804

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
OsDDM1b / OsDDM1R5 / GCAGGTAGACAAGGATATTATGTTCT / RT / this study
OsDDM1F4 / CATCGCTCGCTGCGCACCTCAG / PCR / this study
OsDDM1R4 / ACAGATTAGATGCACACTACCTTATC / PCR / this study
Os07g0431000 / ddmT7-1F1 / ATACATATGTGTGGCCTTCCATGCA / RT / this study
ddmT7-1F2 / TGACTTCACTTCCCATCTGACAACT / PCR / this study
ddmT7-1R2 / GACACATTGGATAGTGCTATGGCCT / PCR / this study
Os12g0428300 / 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
Os11g0677100 / 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
Os06g0493000 / ddmT6-1F1 / TGTCAGGATCCAACGATTTTGGTTC / RT / this study
ddmT6-1F2 / GCTAGTAGCTAACTAATGGGAATCC / PCR / this study
ddmT6-1R2 / GAATTGCCGTACTGTGGACGTCAT / PCR / this study
nDart1-2 / 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) / 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
DaiZ9 / 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
/ RILN39-F1 / GGGATTACCTCCTAGAACTCATGCG / probe for Southern / this study
RILN39-R1 / CCTTGAAGCCGGGTGAGGTTACCTT / probe for Southern / this study
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

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.
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.
Supplementary Table 3. Numbers of transposon-related loci on the 44k microarray and activated loci in the asDDM1 line.
type of transposon-related loci / number of probes on the 44k microarray / number of loci up-regulated in 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
* ≥10 fold increase in the asDDM1 line


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.

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.

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