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Supplementary Information (Molecular Breeding)
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Epigenetic modification in rice controls meiotic recombination and segregation distortion
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Yoshiki Habu, Tsuyu Ando, Sachie Ito, Kiyotaka Nagaki, Naoki Kishimoto, Fumio
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Taguchi-Shiobara, Hisataka Numa, Katsushi Yamaguchi, Shuji Shigenobu, Minoru Murata,
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Tetsuo Meshi, Masahiro Yano
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Corresponding author: Yoshiki Habu, Agrogenomics Research Center, National Institute
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of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba 305-8602, Japan. Phone: +81 29
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838 7442, Fax: +81 29 838 7073, E-mail: habu@affrc.go.jp.
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Supplementary Discussion
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Characterization of the asDDM1 line
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Locations of T-DNAs in the genome of asDDM1 (line 17) were determined by
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whole-genome sequencing using a mate-pair library. Two positions on chromosome 4
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(29.4 Mb and 33.6 Mb positions; Supplementary Fig. S3) were mapped for T-DNA. These
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sites are at the 3’ end of a putative gene (Os04g0573200/ LOC_Os04g48410 at 29.4 Mb
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position) and at around a putative gene (Os04g0649900/LOC_s04g55640 at 33.6 Mb
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position), respectively. Since cytosine demethylation in the centromeric repeat was
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observed in independent transgenic lines producing the same antisense RNA (Higo et al.
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2012), it is unlikely that the disrupted genes in line 17 are responsible for the observed
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hypomethylation. The asDDM1 plants show dwarf phenotypes (Higo et al. 2012) and
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fertility of the F1 (asDDM1 x KS) (57.0%) was lower than that of wild-type F1 (NB x KS)
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(68.4%).
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Alteration in recombination frequency in progenies of F1 (asDDM1 x KS) and
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TSA-treated F1 (NB x KS)
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In the analysis of recombination frequency of chromosome 3, we noted that a considerable
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frequency of meiotic recombination was observed, even in wild-type plants, within a
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region reported as being recombination-repressed in a previous study (Fig. 1b)
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(Harushima et al. 1996). In contrast to the previous study which analyzed progenies of
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field-grown rice plants (Harushima et al. 1996), in this study all the plants including
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wild-type were grown under controlled conditions in a greenhouse, and thus differences in
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growth conditions might explain the observed discrepancies in the position and frequency
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of meiotic recombination in progenies of untreated F1 (NB x KS).
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In addition to chromosome 3, we also analyzed the frequency of meiotic
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recombination in chromosomes 2 and 11. No recombinant was obtained in the analyzed
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region of chromosome 2 between markers RM5210 and RM3443 that are separated by 16
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BACs spanning a sequence gap of unknown size (data not shown). No significant change
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in recombination frequency was also observed in the centromeric region of the
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chromosome 11 (Supplementary Table S1), but an alteration in the distribution of meiotic
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recombination was detected (Supplementary Fig. S4). However, in contrast to the changes
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observed in chromosome 3 (Fig. 1b), the positions of meiotic recombination in the
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centromeric region of chromosome 11 were moved outward from the centromeric core in
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both TSA-treated F1 (NB x KS) and F1 (asDDM1 x KS). Consistent with a previous study
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on Arabidopsis thaliana (Perrella et al. 2010; Colomé-Tatché et al. 2012;
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Melamed-Bessudo and Levy 2012; Mirouze et al. 2012; Yelina et al. 2012), our results
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indicate that the effect of changes in epigenetic state of chromatin on meiotic
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recombination is chromosome-specific.
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Possible non-epigenetic effects of DDM1-knockdown on the changes in the position of
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meiotic recombination and the pattern of segregation distortion
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Reduction in DNA methylation in the genome of Arabidopsis thaliana induces activation
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of transposons (Tsukahara et al 2009; Fu et al 2013). However, Colomé-Tatché et al
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(2012) suggested that frequency of transposition of endogenous TEs in ddm1 of
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Arabidopsis thaliana is kept at low and therefore the authors concluded that the individual
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F1 plants used in the study could be considered homozygous throughout the genome. In
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rice, although treatment with 5-aza-2’-deoxycytidine was shown to activate excision of
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endogenous DNA-type transposons (nDaiZ9 and nDart) (Huang t al 2009; Eun et al 2012),
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our previous study (Higo et al 2012) on the effect of DDM1-knockdown in rice indicated
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that nDaiZ9 and nDart stayed in silent states in asDDM1. In addition, our data of
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genome-wide resequencing of asDDM1 suggested similar low frequencies of transposition
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of endogenous TEs in asDDM1 (data not shown). Large-scale rearrangement of
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chromosome 3 in asDDM1 was not detected in the resequencing data by using three
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independent algorisms for InDel analysis (data not shown). We cannot exclude other
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non-epigenetic (genetic) changes such as spontaneous genome rearrangements in
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individual asDDM1 and/or F1 plants might affect the pattern of segregation distortion.
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However, because the F2 progenies were obtained from four independent F1 plants
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(Materials and Methods), contribution of spontaneous genetic events to the observed
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changes seems to be unlikely. In addition, possible effects of different growth conditions
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on the observed phenomena can be excluded in our experiments (Materials and Methods).
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Concerning TSA-induced changes in the position of meiotic recombination and the
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pattern of segregation distortion, TSA has been used widely as a chemical regulator for
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inducing changes in histone modification in plants and animals (Lawrence et al 2004;
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Taddei et al 2005). Since similar changes in the shift of meiotic recombination position
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(Supplementary Fig. S4) and the pattern of segregation distortion (Fig. 2) were observed
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in asDDM1 and TSA-treated wild-type, the possibility in which spontaneous
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non-epigenetic events caused the similar changes in both asDDM1 and TSA-treated
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wild-type looks highly unlikely.
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Supplementary Methods
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Detection of the positions of T-DNA insertions in line 17
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A mate-pair library (consisting of 0.8–1 kb fragments) was prepared with a SOLiD Long
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Mate Paired Library Enzyme Kit by following the manufacturer’s instructions (Applied
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Biosystems). Data were mapped to a reference genome (The IRGSP genome sequence
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build 5, http://rapdb.dna.affrc.go.jp/) by using SHRiMP2 (David et al. 2011). Pairs
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containing reads corresponding to the T-DNA sequence were picked up and their paired
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non T-DNA reads were mapped to the genome.
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Supplementary References
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Colomé-Tatché M, Cortijo S, Wardenaar R et al (2012) Features of the Arabidopsis
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recombination landscape resulting from the combined loss of sequence variation and
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DNA methylation. Proc Natl Acad Sci USA 109:16240-16245
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David M, Dzamba M, Lister D, Ilie L, Brudno M (2011) SHRiMP2: sensitive yet practical
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Eun CH, Takagi K, Park KI, Maekawa M, Iida S, Tsugane K (2012) Activation and
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epigenetic regulation of DNA transposon nDart1 in rice. Plant Cell Physiol
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53:857-868
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Fu Y, Kawabe A, Etcheverry M, Ito T, Fujiyama A, Colot V, Tarutani Y, Kakutani T (2013)
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of a high frequency transposon induced by tissue culture, nDaiZ, a member of the hAT
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(2004) A concerted DNA methylation/histone methylation switch regulates rRNA
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Paszkowski J (2012) Loss of DNA methylation affects the recombination landscape
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Supplementary Figure Legends
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Supplementary Fig. S1 Characterization of asDDM1, F1 (asDDM1 x KS), and
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TSA-treated F1 (NB x KS). a. Accumulation of centromeric repeat transcripts in wild-type
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and asDDM1. Total RNA was reverse transcribed with forward or reverse primers for
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RCS2 centromeric repeat. ACTIN was used as a control. b,c. Reduction in cytosine
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methylation in centromeric repeats. Southern blot analysis of F1 (asDDM1 x KS) (c) and
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TSA-treated F1 (NB x KS) (d) was done with HhaI and the RCS2 centromeric repeat
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probe (Nonomura and Kurata 2001).
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Supplementary Fig. S2 Changes in RNA accumulation of genes around centromeric
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regions where positions of meiotic recombination were altered in F1 (asDDM1 x KS).
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Average fold expression of genes in asDDM1 relative to that of the wild type is shown. a,
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chromosome 3; b, chromosome 11.
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Supplementary Fig. S3 Locations of T-DNA insertions in asDDM1 determined by
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whole-genome sequencing. Mate-pair reads obtained in whole-genome sequencing of
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asDDM1 were searched for those carrying the sequence of T-DNA. Mate-pairs carrying a
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read for T-DNA and another read for the rice genome sequence (anchored pairs) were
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picked up, and locations of non-T-DNA reads were plotted. Two clusters of the anchored
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pairs were detected on chromosome 4 (around 29.4 Mb [a] and 33.6 Mb position [b]).
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Regions covered by more than 20 reads are indicated with blue.
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Supplementary Fig. S4 Changes in positions of meiotic recombination in the centromeric
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region on chromosome 11. Frequencies in positions of meiotic recombination around the
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centromeric region in chromosome 11 are shown as Fig. 1b.
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