IC4R011-Epigenomic-2015-11001567

Project Title

• Epigenomic modification in rice controls meiotic recombination and segregation distortion

Plant Culture & Treatment

• F1 plants were obtained by crosses between wildtype NB or asDDM1 (female) and KS (male), and grown in a greenhouse. The effects of DDM1 knockdown and TSA treatment on chromatin modification were confirmed by Southern blot analysis with a probe for centromeric repeats (Supplementary Fig. S1b and c). Four F1 plants for each sample [F1 (NB 9 KS),TSA-treated F1 (NB 9 KS) and F1 (asDDM1 9 KS)]were grown in a greenhouse and self-pollinated. Five panicles were taken from each F1 plant, and 15 seeds per panicle were sown in soil. A total of 190 plants[(three F1 plants 9 5 panicles 9 10 seeds) ? (one F1 plant 9 4 panicles 9 10 seeds) = 190] were selected randomly, and genomic DNA was extracted. Genotyping of the F2 progenies was done by PCR using primers shown in Supplementary Table S3. Germination rates were at least 91.3 % for F2 (NB 9 KS),93.6 % for TSA-treated F2 (NB 9 KS) and 93.6 % for F2 (asDDM1 9 KS). The frequencies of morphologically aberrant plants (albino, striped leaf and pale green leaf) were 1.3 % for F1 (NB 9 KS), 0.7 % for TSA-treated F1 (NB 9 KS) and 3.0 % for F1(asDDM1 9 KS). These plants were not used for further analysis.
• F1 seeds were sterilized with 2.5 % sodium hypochlorite for 20 min, washed three times with sterile water and imbibed in sterile water or 100 lM TSA (Sigma)at 30 �C overnight. Imbibed seeds were washed three times with sterile water and grown on Murashige– Skoog medium/0.4 % agar at 30 �C for 7–10 days without TSA. Seedlings were then transferred to soil in a greenhouse and grown further to obtain self-pollinated seeds.

Research Findings

• The rice genome contains two genes homologous to DDM1, and downregulation of two DDM1 genes in a japonica cultivar Nipponbare (NB)by an antisense RNA induced stable cytosine hypomethylation in the centromeric repeat (Higo et al.2012). Accumulation of sense and antisense centromeric transcripts was not apparently changed in the antisense knockdown line of DDM1 (asDDM1) (Supplementary Fig. S1a), indicating that hypomethylation of centromeric repeats does not induce transcriptional activation in the centromeric repeat. as DDM1 was crossed by wild-type Kasalath (KS), which belongs to the aus subpopulation of Oryza sativa L., and the methylation states of the centromeric repeats in the F1 plants were analyzed by methylation-sensitive Southern hybridization (Supplementary Fig. S1b).Variation in methylation in centromeric repeats was observed among independent F1 plants, and this indicates that the recovery of methylation is a stochastic event. The second approach that we took for modifying the epigenetic state of the rice genome was treatment of wild-type F1 seeds with trichostatin A (TSA)—a potent inhibitor of histone deacetylases. TSA induced reduction in methylation in the centromeric repeats,although the effect of TSA varied among samples probably due to variation in TSA penetration into imbibing seeds (Supplementary Fig. S1c).

• An elevation in recombination frequency was observed in a chromosomal interval containing the centromere in the progenies of F1 (asDDM1 9 KS),whereas no significant changes in recombination frequency were detected in the progenies of TSAtreated F1 (NB 9 KS) (Fig. 1a and Supplementary Table S1). In the progenies of F1 (asDDM1 9 KS),locations of increased recombination were restricted

to a region flanking a sequence gap that corresponds to the center of the centromere (Fig. 1b). A recombination hotspot observed in the progenies of F1 (NB 9 KS) (between D7722 and D5711) disappeared in the progenies of F1 (asDDM1 9 KS). Shifts in the position of recombination toward the center of the centromere were also observed in the progenies of TSA-treated F1 (NB 9 KS) (Fig. 1b). We further homed in on the sites of increased recombination in progenies of F1 (asDDM1 9 KS) and found that increased recombination is restricted to a hotspot in the recombination-repressed region (Fig. 1c).

Labs working on this Project

• Agrogenomics Research Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2,Tsukuba 305-8602, Japan
• Institute of Society for Techno-Innovation of Agriculture,Forestry, and Fisheries, Tsukuba 305-0854, Japan
• Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan

Corresponding Author

• Yoshiki Habu:habu@affrc.go.jp