Twenty-four–nucleotide siRNAs produce heritable PNAS PLUS trans-chromosomal methylation in F1 Arabidopsis hybrids Ian K. Greavesa, Steven R. Eichtenb, Michael Groszmanna,b, Aihua Wanga, Hua Yinga,c, W. James Peacocka,d,1, and Elizabeth S. Dennisa,d aCommonwealth Scientific Industrial Research Organization Agriculture and Food, Canberra, Australian Capital Territory (ACT) 2601, Australia; bDivision of Plant Sciences, Research School of Biology Sciences, Australian National University (ANU), Canberra, ACT 0200, Australia; cDivision of Evolution, Ecology and Genetics, Research School of Biology, ANU, Canberra, ACT 0200, Australia; and dUniversity of Technology Sydney, Sydney, NSW 2007, Australia Contributed by W. James Peacock, August 23, 2016 (sent for review June 10, 2016; reviewed by Ryan Lister, Stephen P. Moose, and Stefan Scholten) Hybrid Arabidopsis plants undergo epigenetic reprogramming pro- The TCM and TCdM events do not always occur in the early cell ducing decreased levels of 24-nt siRNAs and altered patterns of divisions of the developing embryo (9). A TCM event overlapping DNA methylation that can affect gene expression. Driving the changes At3g43340 and At3g43350 results in a decrease in expression of in methylation are the processes trans-chromosomal methylation both genes and is stably maintained through successive generations (TCM) and trans-chromosomal demethylation (TCdM). In TCM/TCdM where it continues to correlate with altered levels of gene expression the methylation state of one allele is altered to resemble the other (9, 10). The TCM characteristics defined at At3g43340/50 may not allele. We show that Pol IV-dependent sRNAs are required to establish be representative of how all TCM events are established in the C24/ TCM events. The changes in DNA methylation and the associated Ler F1 hybrid system. A more comprehensive approach is required to changes in sRNA levels in the F1 hybrid can be maintained in sub- assess the impact of TCM/TCdM across the entire C24/Ler F1 hybrid sequent generations and affect hundreds of regions in the F2 epige- genome. In this paper, we determine when the TCM/TCdM events nome. The inheritance of these altered epigenetic states varies in F2 occur during development and if they are inherited into the F2. We individuals, resulting in individuals with genetically identical loci dis- examine what impact these events have on gene expression and the playing different epigenetic states and gene expression profiles. The importance of sRNAs in initiating the TCM/TCdM events. Un- PLANT BIOLOGY change in methylation at these regions is associated with the presence derstanding of the key mechanisms underlying these altered epige- of sRNAs. Loci without any sRNA activity can have altered methylation netic states is essential for determining what impact they may have states, suggesting that a sRNA-independent mechanism may also con- on the hybrid vigor phenotype. tribute to the altered methylation state of the F1 and F2 generations. Results heterosis | gene expression | hybrid vigor | DNA methylation | TCM/TCdM Events Influence mC States in Subsequent Generations. TCM- RNA-dependent DNA methylation mediated mC patterns at the At3g43340/50 locus are inherited into the F2 generation (9). We examined the ability of altered mCstates ybrid vigor, heterosis, is important in agriculture due to the to be retained in subsequent generations on a genome-wide scale Hincreased performance of hybrid plants over parental lines. by profiling parental, F1, and F2 individuals via whole genome bisulphite sequencing (11, 12). There are 17,382 mCG, 3,149 The increase in performance is attributable to changes in gene m m expression (1, 2) but how these changes result in the increased CHG, and 8,270 CHH differentially methylated regions (DMRs) performance of the hybrid is still not known. The level of hybrid vigor is believed to correlate with the genetic distance between Significance parental lines; however, hybrid vigor can occur in progeny de- rived from crosses of genetically similar parents. An example is We show that the changes in DNA methylation that occur in F1 found in intraspecific crosses between Arabidopsis thaliana ac- hybrids of Arabidopsis are mostly dependent on the presence cessions. In crosses between C24 and Landsberg erecta (Ler), up of 24-nt siRNAs at the locus. The methylation change at a locus to a 100% increase in plant biomass and seed yield is observed results in the two alleles becoming similar to each other in (3). The different epigenomes of the two parental accessions methylation pattern. The methylation changes occur through could provide the diversity required to initiate gene expression the processes of trans-chromosomal methylation and trans- changes and vigor-related characteristics. A number of labora- chromosomal demethylation. These altered methylation states tories have investigated whether there are epigenetic changes in can be inherited in the F2 generation and can be associated with changes in levels of gene activity, which may contribute Arabidopsis hybrids, whether they affect gene expression, and to the phenotypic heterogeneity in the F2. whether they contribute to the hybrid vigor phenotype (1, 3–6). Arabi- We have previously established that F1 hybrids between Author contributions: I.K.G., M.G., W.J.P., and E.S.D. designed research; I.K.G. and A.W. dopsis accessions undergo some epigenetic reprogramming. We performed research; I.K.G., S.R.E., and H.Y. analyzed data; and I.K.G., W.J.P., and E.S.D. found that the abundance of 24-nt siRNAs is reduced in F1 hybrids wrote the paper. m and that a second epigenetic system, DNA methylation ( C), is also Reviewers: R.L., University of Western Australia; S.P.M., University of Illinois; and S.S., altered in the hybrid genome (1, 3, 5). The altered mCpatternsinthe Institute of Plant Breeding, Seed Science, and Population Genetics, University of Hohenheim. hybrids involve the processes trans-chromosomal methylation (TCM) and trans-chromosomal demethylation (TCdM), where the mC state The authors declare no conflict of interest. of one parental allele is altered to resemble the other allele (reviewed Freely available online through the PNAS open access option. in ref. 7). Due to the presence of siRNAs at regions that undergo Data deposition: The data including raw sequencing files and processed files reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www. TCM/TCdM, we have suggested that siRNAs are the initiating ncbi.nlm.nih.gov/geo (accession no. GSE85551). molecules that establish these TCM/TCdM events (reviewed in ref. 1To whom correspondence should be addressed. Email: [email protected]. 8). In the hybrid nucleus, the complement of 24-nt siRNAs can in- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. teract with homologous sequences from either parent. 1073/pnas.1613623113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1613623113 PNAS Early Edition | 1of8 Downloaded by guest on September 27, 2021 Fig. 1. Inheritance of TCM/TCdM-mediated mC states. (A)ThemC levels of F2-inherited windows in two time points of F1 hybrid development. F2.C24, inherited windows homozygous for C24 in the F2 individuals (see SI Appendix, Fig. S2 for F2.Ler); 15 DAS, mC levels in F1 15 d whole seedlings; F1.buds, mC levels in F1 hybrid immature inflorescence; and F2.buds, mC levels in F2 immature inflorescence. Dotted green line represents MPV. The * above F1.buds represents a statistical difference in normalized mC levels between F1 15 DAS and F1 buds (P ≤ 0.01; ANOVA test). NS, normalized mC levels between F1 15 DAS and F1 buds that are not statistically significant. (B) McrBC qRT-PCR was used to analyze mC levels at several time points of the F1 hybrid life cycle. (C) Total windows (green) and inherited windows (red) were mapped to different genomic features for mCG. Features were split into those overlapping TEs (TE.associated) and features not associated with TEs. Features mapped to include intragenic (GeneBody), TE genes (TEG), pseudogenes (Pseudo), 1 kb upstream, 1 kb downstream, and intergenic. (D) Association of total DMRs and inherited windows with TEs. χ2 test, *P ≤ 0.01. (H represents A, C, or T) between the two parental accessions that We found that the TCM event at At3g43340/50 had not occurred could potentially undergo TCM/TCdM in the F1 and be tracked at 15 days after sowing (DAS) but was present in the immature into the F2 generation (Table 1 and SI Appendix, SI Materials inflorescence of F1 hybrids (9). We suggested that TCM events may and Methods). occur due to a build-up of an inducing molecule that triggers a TCM Individual F2 plants were genotyped and chromosomal seg- event (reviewed in refs. 8 and 13). To test this on a genome-wide ments defined as being homozygous C24, homozygous Ler,or scale, we compared mC levels of the F2-inherited windows at two SI Appendix A m heterozygous ( , Table S1 and Fig. S1 ). C levels of time points in the F1 hybrid, 15 DAS and immature inflorescence m homozygous regions of the F2 were compared with the C level (buds; Fig. 1A). The mCG level at these regions increased from the of the parent of origin for that segment. If the segment in the F2 earlier time point (15 DAS) to the later time point (immature had mC levels different from the parent of origin, it was assumed to have inherited a TCM/TCdM-mediated mC pattern from the F1 hybrid (SI Appendix, Fig. S1B and SI Materials and Methods). Table 1. Differentially methylated regions m m In the F2 individuals, there were 542 CG, 450 CHG, and 4,964 Methylation Methylation Differentially m m CHH DMRs that differed in C levels from the parent of or- level above level below methylated igin (Table 1 and SI Appendix, SI Materials and Methods).