Positional Cloning,Epigenetic Modification And Functional Analyses Of A Dominant Dwarf Gene Of Epi-df In Rice(Oryza Sativa L.)

Plant height is an agronomically important trait for grain yield, the higher plant is easy to be lodging and decreasing yield, whereas dwarf plant has a great harvest index because of improved lodging resistance and increasing use of nitrogen fertilizers. Dwarfism breeding resulted in the first qualitative leap for yield increase, which was well known as "green revolution". The objective of super rice breeding is to make a breakthrough in rice yield by using ideotype and inter-subspecific heterosis. Whereas all the dwarf genes used in rice breeding is recessive by now, thus both the male and female parents must carry the same recessive dwarf gene in hybrid rice breeding process. But when one parent carries a dominant dwarf allele, the germplasm of the other parent would not be restricted. Therefore, isolation new genes that control plant height especially the dominant dwarf genes is crucial for improving rice yield. In this study, we systematacially characterized a dominant dwarf mutant Epi-df and analyzed the molecular mechanism for dwarfism.The main results as follows:(1) From seedling to mature stage, Epi-df showes dwarf phenotype. When crossed with WT, the F1plants show the phenotype similar to Epi-df, thus, Epi-df is a dominant dwarf mutant. When crossed with PA64, the plant height of F1is between two parents, and the ratio of normal to dwarf plants in F2population is nearly to1:3, suggesting that the mutant phenotype is controlled by one dominant nuclear gene. When Epi-df was crossed with three indica varieties and three japonica varieties respectively, the plant height reduction is from16.3to50.4%, suggesting the strong plant height reduction ability. Thus, Epi-df may be used for rice inter-subspecific heterosis breeding in the future.(2) The mutative gene was fine mapped within a49kb region, whereas there was no nucleotide mutation. However, we found ORF5(one of the seven ORFs within the mapping region) was ectopically expressed in Epi-df, but silenced in WT. Considered the revertants emerged from Epi-df population, we speculated that Epi-df was an epigenetic mutant. By using bisulfite sequencing, we found DNA hypo-methylation was occurred at5’region of FIE1. We also analyzed the methylation patterns of six revertants and found even FIE I was silenced in them, DNA methylation was not recovered to the WT level, the recovered sits were enriched around the transcriptional starting site and within the third exon. We also found there were reduced H3K9me2and increased H3K4me3at the5’region of FIE1. Thus, Epi-df is an unexpected epigenetic mutant, which would like to provide an intriguing opportunity to unravel epigenetic modifications for development regulation in important crop plants.(3) We discovered that FIE1was a maternal-specific expressing gene in endosperm, which was coincided with the methylation pattern of FIE1in tissues. The methylation level is higher in leaf, culm and young panicle than that in endosperm6,9and12days after pollination. We also found the methylation of maternal FIE1was much lower than that of paternal. If Epi-df was used as pollen donator, the imprinting pattern was disturbed, and the methylation levels of both parental were lower. Unlike Arbidopsis, which contains only one ubiquitously expressed FIE gene, there are two FIE gene in rice, the other gene FIE2is expressed in all the tissues. We conclude that during the genome duplication and the latter evolution, epigenetic marks may play an important role in the differentiation of the two FIE gene in rice.(4) Yeast two-hybrid assay showed FIE1interacted with rice E(z) homologs, suggesting FIE1participates in PRC2repression which catalyzes H3K27me3at targets. Microarray analysis showed305genes were misregulated in Epi-df accompanied with changed H3K27me3levels. Thus, ectopic expression of FIE1resulted in the mutant phenotype via abnormal distribution of H3K27me3.(5) H3K9me2and H3K27me3are two conserved repressive epigenetic marks in both animal and higher plants. H3K9me2is mainly enriched in heterochromatin and functions in suppressing transposons, while H3K27me3is mainly localized in euchromatin and provides a cellular memory to maintain the repressive state of target genes. We found there was high level of H3K9me2at5’region of FIE1, whereas in Epi-df, H3K9me2was reduced and resulted in ectopic expression of FIE1and abnormal distribution of H3K27me3. We conclude that silencing of FIE1via H3K9me2is essential for normal function of H3K27me3in rice.