Parental DNA methylation states are associated with heterosis in epigenetic hybrids

69 Pages·2017·2.24 MB·English

Plant Physiology Preview. Published on December 1, 2017, as DOI:10.1104/pp.17.01054

1 Short title: Epigenetic divergence associated with heterosis
2 § Corresponding co-last authors:
3 Maike Stam, University of Amsterdam, Swammerdam Institute for Life Sciences, Science
4 Park 904 1098XH Amsterdam, The Netherlands.
5 Frank Johannes, Population epigenetics and epigenomics, Department of Plant Sciences,
6 Technical University of Munich, Liesel-Beckmann-Str. 2, 85354 Freising, Germany
7
8 Parental DNA methylation states are associated with heterosis in
9 epigenetic hybrids
10
11
12 Kathrin Lauss1*, René Wardenaar2*, Rurika Oka1, Marieke H.A. van Hulten3, Victor Guryev4,
13 Joost J.B. Keurentjes3, Maike Stam1§, Frank Johannes5,6§
14
15 1 University of Amsterdam, Swammerdam Institute for Life Sciences, Science Park 904
16 1098XH Amsterdam, The Netherlands.
17 2 University of Groningen, Groningen Bioinformatics Centre, Faculty of Mathematics and
18 Natural Sciences, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
19 3 Wageningen University & Research, Laboratory of Genetics, Droevendaalsesteeg 1,
20 6708PB Wageningen, The Netherlands.
21 4 Genome structure aging, European Research Institute for the Biology of Ageing, University
22 Medical Centre Groningen and University of Groningen, Antonius Deusinglaan 1, Building
23 3226, 9713 AV Groningen, The Netherlands
24 5 Population epigenetics and epigenomics, Department of Plant Sciences, Technical
25 University of Munich, Liesel-Beckmann-Str. 2, 85354 Freising, Germany
26 6 Institute for Advanced Study, Technical University of Munich, Lichtenbergstr. 2a, 85748
27 Garching, Germany
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28 * co-first authors
29 § co-last authors
30
31 One sentence summary:
32 DNA methylation differences between isogenic parental lines can directly or indirectly
33 trigger heterosis in Aradidopsis hybrids.
34
35 Author Contributions
36 K.L., M.S. and F.J. designed the study, interpreted the data and wrote the manuscript with
37 contributions from J.J.B.K. and R.W. K.L. and M.H.A.v.H. planned and performed the
38 phenotypic screen. R.W., K.L., V.G., F.J. and R.O. performed data analysis.
39
40 Funding
41 We acknowledge the support of the Centre for Improving Plant Yield (CIPY; part of the
42 Netherlands Genomics Initiative and the Netherlands Organization for Scientific Research)
43 for Kathrin Lauss. The first phenotypic screen was supported by the Enabling Technologies
44 Hotels (ETH) Programme from CIPY. We gratefully acknowledge the financial support of the
45 European Commission 7th Framework-People-2012-ITN Project EpiTRAITS (Epigenetic
46 Regulation of Economically Important Plant Traits; 316965) to Rurika Oka and Maike Stam.
47 Frank Johannes acknowledges support from the Technical University of Munich-Institute for
48 Advanced Study funded by the German Excellence Initiative and the European Union
49 Seventh Framework Programme under grant agreement #291763, and the
50 SFB924/Sonderforschungsbereich924 of the Deutsche Forschungsgemeinschaft (DFG).
51
52 Corresponding co-last authors: [email protected] (MS), [email protected] (FJ)
53
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54 Abstract
55 Despite the importance and wide exploitation of heterosis in commercial crop breeding, the
56 molecular mechanisms behind this phenomenon are not completely understood. Recent
57 studies have implicated changes in DNA methylation and small RNAs in hybrid performance,
58 however, it remains unclear whether epigenetic changes are a cause or consequence of
59 heterosis. Here, we analyze a large panel of over 500 A. thaliana epigenetic hybrid plants
60 (epiHybrids), which we derived from near-isogenic but epigenetically divergent parents. This
61 proof-of-principle experimental system allowed us to quantify the contribution of parental
62 methylation differences to heterosis. We measured traits such as leaf area (LA), growth rate
63 (GR), flowering time (FT), main stem branching (MSB), rosette branching (RB) and final plant
64 height (HT) and observed several strong positive and negative heterotic phenotypes among
65 the epiHybrids. Using an epigenetic quantitative trait locus mapping approach, we were able
66 to identify specific differentially methylated regions (DMRs) in the parental genomes that
67 are associated with hybrid performance. Sequencing of methylomes, transcriptomes and
68 genomes of selected parent-epiHybrid combinations further showed that these parental
69 DMRs most likely mediate remodeling of methylation and transcriptional states at specific
70 loci in the hybrids. Taken together, our data suggest that locus-specific epigenetic
71 divergence between the parental lines can directly or indirectly trigger heterosis in
72 Arabidopsis hybrids independent of genetic changes. These results add to a growing
73 body of evidence that points to epigenetic factors as one of the key determinants of
74 hybrid performance.
75
76 Introduction
77 Heterosis describes an F1 hybrid phenotype that is superior compared to the phenotype of
78 its parents. The phenomenon has been exploited extensively in agricultural breeding for
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79 decades and has improved crop performance tremendously (Chen, 2010; Schnable and
80 Springer, 2013). Despite its commercial impact, knowledge of the molecular basis underlying
81 heterosis remains incomplete. Most studies have focused on finding genetic explanations,
82 resulting in the classical dominance (Jones, 1917; Crow, 1998; Schnable and Springer, 2013)
83 and overdominance (Crow, 1948; Crow, 1998) models of heterosis. In line with genetic
84 explanations, interspecies hybrids have been frequently observed to show a higher degree
85 of heterosis than intraspecies hybrids, indicating that genetic distance correlates with the
86 extent of heterosis (East, 1936; Chen, 2010).
87 Genetic explanations do not, however, sufficiently explain nor predict heterosis. There is
88 growing evidence that also epigenetic factors play a role in heterosis (Groszmann et al.,
89 2013; Springer, 2013; Dapp et al., 2015a; Zhu et al., 2017). It has, for example, been shown
90 that altered epigenetic profiles at genes regulating circadian rhythm play an important role
91 in heterotic Arabidopsis hybrids (Ni et al., 2009a). Moreover, heterotic hybrids of
92 Arabidopsis, maize and tomato are shown to differ in levels of small regulatory RNAs and/or
93 DNA methylation (5mC) relative to their parental lines (Groszmann et al., 2011; Barber et al.,
94 2012; Shen et al., 2012; Shivaprasad et al., 2012; Zhang et al., 2016b).
95 Remodeling of the methylome during hybridization has been proposed to be involved in
96 heterosis in genetic hybrids, and was implicated in the formation of novel epialleles in a
97 met1-derived epiHybrid (Groszmann et al., 2011; Greaves et al., 2012; Shen et al., 2012;
98 Rigal et al., 2016). Processes such as the transfer of 5mC between alleles (trans-
99 chromosomal methylation, TCM), or a loss of 5mC at one of the alleles (trans-chromosomal
100 demethylation, TCdM) have been indicated to contribute to the observed remodeling of the
101 epigenome (Greaves et al., 2012; Shivaprasad et al., 2012; Groszmann et al., 2013). These
102 TC(d)M events occur between homologous sequences and have been shown to require the
103 RNA-directed DNA methylation (RdDM) pathway, which involves small regulatory RNAs
104 (Zhang et al., 2016b). Strikingly, some of these changes in 5mC levels have been shown to be
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105 stable over multiple generations (Greaves et al., 2012; Greaves et al., 2014). However,
106 parental lines used for these studies, differed in both their genetic and epigenetic profile,
107 making it challenging to disentangle genetic from epigenetic effects.
108 It has been shown in isogenic epigenetic recombinant inbred lines (epiRILs) that
109 heritable morphological variation in Arabidopsis plants can be exclusively caused by
110 epigenetic factors (Johannes et al., 2009; Roux et al., 2011; Cortijo et al., 2014; Kooke and
111 Keurentjes, 2015). To specifically address the contribution of parental epigenetic variation to
112 F1 heterosis we made use of the same epiRILs (Johannes et al., 2009; Reinders et al., 2009)
113 to generate F1 epigenetic hybrids, hereafter called epiHybrids (Dapp et al., 2015a). EpiRILs
114 are near isogenic, but display mosaic patterns in terms of their epigenomes. The two
115 Arabidopsis epiRIL populations reported have been created by crossing the wild-type
116 Columbia accession (Col-wt) with Col-wt lines carrying a mutation in either
117 METHYLTRANSFERASE 1 (MET1-3) or DECREASE IN DNA METHYLATION 1 (DDM1–2)
118 (Johannes et al., 2009; Reinders et al., 2009). MET1 maintains DNA methylation at cytosines
119 in a CG sequence context (mCG), but also affects non-CG methylation (Finnegan et al., 1996;
120 Mathieu et al., 2007; Stroud et al., 2013). Loss of MET1 causes an almost complete
121 elimination of mCG genome-wide, which is associated with misregulated gene expression
122 and transcriptional activation of TEs (Zhang et al., 2006; Cokus et al., 2008; Lister et al.,
123 2008). DDM1 is a nucleosome remodeler and the ddm1-2 mutation leads to a ~70%
124 reduction in DNA methylation (Kakutani et al., 1995), predominantly affecting mCG and
125 mCHG (where H represents A, C or T), and to a lesser extent mCHH (Zemach et al., 2013), in
126 primarily long transposable elements (Zemach et al., 2013). Loss of DDM1 also affects genic
127 loci where it reduces mCG and causes CHG hypermethylation in gene bodies (Zemach et al.,
128 2013).
129 Recently, epiHybrids have been generated by crossing a met1-derived epiRIL with
130 Col-wt, and heterosis for biomass was reported for one of the epiHybrids (Dapp et al.,
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131 2015a). The effect was only observed with the epiRIL as maternal parent, suggesting a strong
132 maternal effect (Dapp et al., 2015a). In this study we created epiHybrids by crossing Col-wt
133 as a maternal parent to nineteen different near-isogenic but epigenetically divergent ddm1-
134 derived epiRILs, allowing the assessment of epigenetic variation in identical maternal
135 backgrounds. Using high-throughput phenotyping, we observed various positive and
136 negative heterotic effects in one or more of the six traits monitored among the nineteen
137 epiHybrids, indicating that epigenetic divergence among parents has a direct or indirect role
138 in triggering heterosis. Furthermore, in contrast to previous studies, our experimental design
139 allowed us to employ an epigenetic quantitative trait locus mapping approach that, in
140 combination with methylome and transcriptome profiling, allowed quantifying and
141 characterizing the contribution of parental methylation differences to heterosis. Using this
142 approach we were able to identify specific differentially methylated regions (DMRs) in the
143 parental genomes that are associated with heterotic phenotypes in the epiHybrids. We
144 provide evidence that these parental DMRs mediate local methylome and transcriptome
145 remodeling at putatively causative genes, thus providing molecular mechanisms underlying
146 the observed heterotic phenotypes in the epiHybrids.
147
148 Results
149 Construction of epigenetic Hybrids
150 Hybrids are usually generated from parental lines that vary at both the genomic and
151 epigenomic level and disentangling those two sources of variation is challenging. To
152 overcome this limitation, we generated epigenetic A. thaliana F1 hybrids (epiHybrids) from
153 near-isogenic but epigenetically divergent parental lines by crossing Col-0 wildtype (Col-wt)
154 as maternal parent to near-isogenic ddm1-2-derived epigenetic recombinant inbred lines
155 (epiRILs) (Johannes et al., 2009) as the paternal parents (Fig. 1a). EpiRILs carry chromosomes
156 that are a mosaic of Col-wt and hypomethylated ddm1-2-derived genomic regions (Johannes
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157 et al., 2009; Colomé-Tatché et al., 2012; Cortijo et al., 2014) (Fig. 1A). Nineteen epiRIL
158 parental lines were selected that sample a broad range of 5mC divergence from the Col-wt
159 reference methylome (Fig. 1B, Supplemental Table S1). Besides, lines were chosen that have
160 a wildtype methylation profile at FLOWERING LOCUS WAGENINGEN (FWA) (Supplemental
161 Fig. S1, Table S1), as loss of DNA methylation at the FWA locus is known to affect flowering
162 time and, as such, many related developmental traits (Soppe et al., 2000). Furthermore, we
163 selected epiRILs covering a wide range of phenotypic variation in flowering time and root
164 length, two traits that have previously been monitored (Supplemental Table S1) (Johannes et
165 al., 2009).
166
167 Heterotic phenotypes occur in the epiHybrids
168 The phenotypic performance of the 19 epiHybrid lines and their 20 parental lines was
169 assessed. In total, we monitored about 1090 plants (~28 replicate plants per epiHybrid or
170 parental line) for a range of quantitative traits: leaf area (LA), growth rate (GR), flowering
171 time (FT), main stem branching (MSB), rosette branching (RB), plant height (HT) and seed
172 yield (SY) (Supplemental Tables S2-7). The hybrids and parental lines were grown in parallel
173 in a climate-controlled chamber with automated watering. The plants were randomized
174 throughout the chamber to level out phenotypic effects caused by plant position. Leaf area
175 was measured up to 14 days after sowing (DAS), using an automated camera system (Fig.
176 1C), and growth rate was determined based on these data (Supplemental: Supplemental
177 Methods). Flowering time was scored manually as the day of opening of the first flower.
178 After all plants started flowering, they were transferred to the greenhouse and grown to
179 maturity. Main stem branching, rosette branching and height were scored manually after
180 harvesting of the plants. The phenotypic observations for seed yield were inconsistent in a
181 replication experiment, therefore those datasets were excluded from further analysis.
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182 The extent of heterosis was evaluated by comparing the phenotypic performance of
183 the hybrids to that of their parental lines. We distinguished five effects: additivity, positive
184 mid-parent heterosis (positive MPH), negative mid-parent heterosis (negative MPH), high-
185 parent heterosis (HPH) and low-parent heterosis (LPH; Fig. 1D-F). Briefly, an additive effect is
186 defined by a hybrid mean phenotype that is equal or close to the average phenotype of the
187 two parents (the mid-parent value, MPV). Mid-parent heterosis, by contrast, refers to
188 positive or negative deviations of the hybrid mean phenotype from the MPV. High-parent
189 heterosis and low-parent heterosis are important special cases of mid-parent heterosis in
190 which the hybrid mean phenotype either exceeds the mean phenotype of the high parent,
191 or falls below that of the lowest performing parent. In crop breeding, the focus is usually on
192 obtaining high parent heterosis (HPH) and low parent heterosis (LPH) as these present novel
193 phenotypes that are outside the parental range. Depending on the trait and commercial
194 application, either high-parent heterosis or low-parent heterosis can be considered superior.
195 For instance, early flowering may be preferable over late flowering; in such cases maximizing
196 low-parent heterosis may be desirable. For other traits, such as yield or biomass, it is more
197 important to maximize high-parent heterosis. However, in order to obtain a comprehensive
198 view of hybrid performance it is informative to also monitor mid-parent heterosis, as many
199 traits of mature plants are affected by other traits that may not display fully penetrant
200 heterotic effects.
201 We observed a remarkably wide range of heterotic phenotypes among the
202 epiHybrids (Fig. 1G, Supplemental Tables S2-7). The magnitude of these phenotypic effects
203 was substantial (Fig. 1H-J, Supplemental Fig. S2, Tables S8-19) and similar to that typically
204 seen in hybrids of Arabidopsis natural accessions (Groszmann et al., 2014; Wang et al.,
205 2015). Many epiHybrids (16/19) exhibited significant mid-parent heterosis in at least one of
206 the six monitored traits (FDR = 0.05, Fig. 1G). Across all epiHybrids and traits we identified 30
207 cases of positive mid-parent heterosis and negative mid-parent heterosis. Among those, four
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208 cases show low-parent heterosis and nine cases show high-parent heterosis (Fig. 1G).
209 Interestingly, in 11 out of the 17 remaining cases of mid-parent heterosis, the phenotypic
210 means of the epiHybrids were in the direction of the phenotypic means of the epiRIL parent
211 rather than in the direction of the Col-wt parent (Fig. 1G, Supplemental Tables S2-7, F1
212 trend). Also, all four low-parent heterosis and two of the high-parent heterosis cases were in
213 the direction of the epiRIL parent (Fig. 1G, I-J, Supplemental Fig. S2). This observation
214 illustrates that ddm1-2-derived hypomethylated epialleles are often (partially) dominant
215 over wild-type epialleles.
216 We observed cases of high-parent heterosis for leaf area (LA), height (HT) and main
217 stem branching (MSB), and cases of low-parent heterosis for flowering time (FT) and main
218 stem branching. High-parent heterosis for leaf area (LA) occurred in 3 out of 19 epiHybrids,
219 namely 232H, 195H and 193H (the number in the ID refers to the epiRIL parent and the H
220 stands for F1 epiHybrid line). EpiHybrids 232H, 195H and 193H significantly exceeded their
221 best parent (Col-wt) by 17%, 18% and 15%, respectively (Fig. 1H, Supplemental Table S19).
222 Interestingly, although growth rate is developmentally related to leaf area, hybrid effects in
223 growth rate were only moderately, albeit positively, correlated with leaf area (rho = 0.57, P =
224 0.02), which implies that leaf area heterosis is determined by other traits besides growth
225 rate.
226 For height, we detected five cases of significant high-parent heterosis with up to 6%
227 increases in height (Fig. 1I, Supplemental Table S14). One may expect leaf area high-parent
228 heterosis to strongly correlate with height high-parent heterosis, as the rosette is providing
229 nutrients for the developing shoot (Bennett et al., 2012). However, high-parent heterosis for
230 both leaf area and height occurred only in one epiHybrid (193H; Fig. 1G). For main stem
231 branching, we detected one case of high-parent heterosis (64H; Fig. 1G and Supplemental
232 Fig. S2).
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233 Besides positive heterosis, our phenotypic screen revealed strong negative heterotic
234 effects for flowering time (earlier flowering) and main stem branching (less main stem
235 branching). Significant low-parent heterosis occurred in epiHybrids 232H, 208H and 344H
236 (FT) and 438H (MSB) (Fig. 1J, Supplemental Fig. S2, Tables S15 and S17). In the most
237 prominent case for flowering time, the epiHybrid (232H) flowered about 10% earlier than
238 that of the earliest flowering parent. EpiHybrids 208H and 344H flowered 3% and 4% earlier
239 than their earliest parent (epiRIL 208 and epiRIL 344), respectively. EpiHybrid 438H showed
240 14% less main stem branching than its least branched parent (Supplemental Fig. S2).
241 The reproducibility of our findings was tested by performing replicate experiments,
242 using seeds from newly performed crosses under the same growth conditions as before. We
243 focused on epiHybrids that exhibited relatively strong positive or negative heterotic
244 phenotypes in the initial screen (193H, 150H, 232H; Fig. 1G), and measured leaf area,
245 flowering time and height in the epiHybrids and their parents. We monitored about 540
246 plants for leaf area (~60 replicates per line) and 270 plants for flowering time and height
247 (~30 replicates per line). The direction of the heterotic effects in leaf area, flowering time
248 and height was reproducible in all tested cases (Fig. 2A and B). Importantly, the leaf area and
249 height high-parent heterosis observed for 193H, and the strong flowering time low-parent
250 heterosis for 232H were perfectly reproducible, while leaf area high-parent heterosis
251 observed for 232H was reduced to mid-parent heterosis (Fig. 2A). Taken together, these
252 results show that the heterotic effects observed in the epiHybrids are relatively stable for
253 leaf area, height and flowering time even across fresh parental seed batches and
254 independently performed crosses, which is not always the case for Arabidopsis phenotypes
255 (Massonnet et al., 2010).
256
257 Parental DMRs are associated with epiHybrid performance
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