IC4R011-Epigenomic-2015-11001567

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Project Title

  • Epigenomic modification in rice controls meiotic recombination and segregation distortion


The Background of This Project

  • Centromeric heterochromatin in higher plants is characterized by higher levels of DNA methylation and histone H3 lysine 9 methylation (Lister et al. 2008;Bernatavichute et al. 2008). Meiotic recombination is repressed in the centromeric heterochromatin region(Copenhaver et al. 1999; Wu et al. 2003), although a number of active genes have been shown to be present in recombination-repressed centromeric regions in higher eukaryotes (Saffery et al. 2003; Nagaki et al.2004).
  • Recent studies on Arabidopsis thaliana showed that deficiency in the maintenance of chromatin modification leads to changes in the position of meiotic recombination, but its effect on centromeric heterochromatin regions varies (Perrella et al. 2010;Colome ´-Tatche ´ et al. 2012; Melamed-Bessudo and Levy 2012; Mirouze et al. 2012; Yelina et al. 2012).


Plant Culture & Treatment

'Supplementary Fig. S1(b)'
  • 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

  • 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).


'Fig. 1 Changes in recombination frequency and positions in chromosome 3. a Frequency of meiotic recombination in chromosome 3. The frequency of meiotic recombination in each chromosome segment is shown as the frequency relative to that of F2 progenies of untreated F1 (NB 9 KS). Marker positions are shown at the bottom in cM and Mb, and the position of the centromere is indicated with an oval. White bars,F2 progenies of TSA-treated F1 (NB 9 KS); black bars, those of F1 (asDDM1 9 KS). b Changes in positions of meiotic recombination around the centromeric region (between RM1940 [86.0 cM] and C60465 [87.1 cM]) in TSA-treated wild-type F1 (NB 9 KS) and F1 (asDDM1 9 KS). The previously reported CENH3 region (Yan et al. 2005) and recombination-free region reported in a previous study(Harushima et al. 1996) are shown as boxes. c Fine mapping of the recombination hotspot observed in F2 progenies of F1(asDDM1 9 KS). Triangles indicate positions of expressed genes. All of four markers (D3806, YH03182Bg, YH0320 and YH0302) locate within a 86.0 cM region'


  • Consistent with previous studies (Harushima et al.1996), a significant distortion was observed in untreated wild-type F2 progenies at around the 66 cM position on chromosome 3 (Fig. 2). Progenies of the TSA-treated F1 (NB 9 KS) and F1 (asDDM1 9 KS) also showed similar distortion at this site, indicating that inhibition of histone deacetylases and knockdown of the DDM1 activity do not affect distortion at this site. Another distortion was detected at around 160 cM in the untreated wild type (Fig. 2). Although a distortion at this site was also reported in a previous study (Harushima et al. 1996), the pattern of distortion was different from our current data; in the previous work, an increase in the proportion of KS homozygotes and a decrease in the number of heterozygotes were detected. We speculate that this discrepancy might be caused by the difference in the growth condition between two studies (field and greenhouse,respectively) as reported on maize abnormal chromosome 10 in which a heterochromatin knob is a causative agent for the distortion (Rhoades 1942).The distortion at around 160 cM disappeared in the progenies of both TSA-treated F1 (NB 9 KS) and F1(asDDM1x KS) (Fig. 2), indicating that changes in chromatin modification induced by TSA treatment and DDM1 knockdown affect distortion in this region. A striking change in the distortion pattern in F2(asDDM1 9 KS) was detected in a region including the centromere where the frequency of heterozygotes is higher than 70 % at its peak with a concomitant decrease in both NB and KS homozygotes (Fig. 2).


'Fig. 2 Effect of TSA treatment and DDM1 knockdown on patterns of segregation distortion. Genotypes of F2 progenies of F1 (NB 9 KS) (top), TSA-treated F1 (NB 9 KS) (middle) and F1 (asDDM1 9 KS) (bottom) were determined with PCR-based markers on chromosome 3. Percentages of F2 progenies heterozygous (circles), homozygous for NB (squares) and homozygous for KS (triangles) at each position are shown.Arrowheads with asterisks indicate significant segregation distortion detected by v2test (***p\ 0.001; **p\ 0.01;*p\ 0.05). The position of the recombination hotspot shown in Fig. 1c is indicated with gray bars'


  • The researchers performed chromatin immunoprecipitation with antibodies against CENH3 as well as differentially modified N-terminal tails ofhistone H3 and H4 inthe centromeric region of chromosome 3 in wild-type NB and asDDM1.The results showed that deposition of H3K9me2 in the centromeric region depends on the DDM1 activity,whereas the high levels of di-methylated lysine 4 in histone H3 (H3K4me2) are not changed by the DDM1 deficiency (Fig. 3). Acetylated histone H4 (H4Ac) was low in most of the regions analyzed, and this is consistent with a previous observation that H3K4me2-and H4Ac-positive regions do not always coexist in rice centromeric regions (Yan et al. 2005). An enrichment of CENH3 in a control centromeric regiononchromosome 8(Nagakietal.2004)was observed,and thisenrichment was lost in asDDM1 (Fig. 3b).


'Fig. 3 Changes in distribution of CENH3 and histone modifications within the centromeric region in asDDM1. A schematic figure of the analyzed sites (a) and results of quantitative PCR for selected sites (b) are shown. 8C018 is a control CENH3-rich region in chromosome 8 (Nagaki et al. 2004). Site 68 locates within the recombination hotspot detected in the F2 progenies of'


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