Hybrid vigor in plants, and the role of epigenetics.

Hybrid vigor in plants, and the role of
epigenetics
MSc Biology literature study/thesis
2016
Student: C.W.M Rijkenberg
University of Amsterdam (UvA), Faculty of Natural Sciences, Mathematics and Informatics, SILS
Sciencepark 904
1090 GE Amsterdam
The Netherlands

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Index
Abstract ................................................................................................................................................................... - 2 -
Introduction............................................................................................................................................................. - 3 -
Heterosis or hybrid vigor..................................................................................................................................... - 3 -
Importance of hybrid vigor.................................................................................................................................. - 4 -
Scope of the report.............................................................................................................................................. - 5 -
The genetic basis of heterosis ................................................................................................................................. - 6 -
Genetic models.................................................................................................................................................... - 6 -
Dominance....................................................................................................................................................... - 6 -
Over-dominance.............................................................................................................................................. - 7 -
Pseudo overdominance................................................................................................................................... - 8 -
Epistasis ........................................................................................................................................................... - 8 -
Gene balance hypothesis................................................................................................................................. - 9 -
Selective protein synthesis and metabolism hypothesis..................................................................................... - 9 -
Limitations of genetics in explaining heterosis ................................................................................................. - 10 -
Epigenetics and heterosis...................................................................................................................................... - 11 -
Effects of hybridization and allopolyploidization on genome-wide epigenetic and small RNA patterns of
heterotic progeny.............................................................................................................................................. - 13 -
Epigenetic states of genes and transposon elements in hybrids and allopolyploids........................................ - 20 -
Site specific inheritance patterns of epigenetic modifications and small RNAs, in hybrids and allopolyploids- 22 -
Associations between siRNA and DNA methylation inheritance patterns in hybrids....................................... - 24 -
Models for DNA methylation, small RNA, and histone modification inheritance patterns in hybrids ............. - 27 -
Paramutation..................................................................................................................................................... - 29 -
Past F1 loss of hybrid vigor................................................................................................................................ - 29 -
Hybrid mimics (past F1 heterosis) ................................................................................................................. - 30 -
Genetic and epigenetic dynamics at fertilisation in hybrids and allopolyploids............................................... - 31 -
Parent of origin effects.................................................................................................................................. - 32 -
Hybrid incompatibility ................................................................................................................................... - 33 -
Epigenetic pathway mutant studies.................................................................................................................. - 34 -
Altered epigenomes, the circadian clock, and photosynthesis in F1 hybrids and allopolyploids..................... - 35 -
Investigating the role of epigenetics in heterosis: restrictions, assumptions and research methodologies.... - 37 -
Concluding remarks............................................................................................................................................... - 38 -
Methodology ......................................................................................................................................................... - 40 -
References............................................................................................................................................................. - 41 -

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Abstract
Heterosis, or hybrid vigor refers to the phenomenon in which hybrid and allopolyploid progeny of parents that are
genetically diverged to some extent, possess trait characteristics that surpass those exhibited by either parent.
These characteristics are often related to vigor and include biomass, growth rate, (seed) yield, and stress
tolerance. Therefore, hybrid and allopolyploid plants are widely used in horticulture and agriculture. However,
although this phenomenon has been recognized already for a long time, and despite the considerable amount of
research that has been directed at the topic, the molecular basis and mechanisms underlying heterosis remain
largely elusive. Several models based on classical genetics have been proposed to explain hybrid vigor and the
transcriptome alterations that occur in hybrids and allopolyploids, which cause the heterotic plant phenotype.
These models however, have their limitations. Plant, and thus hybrid transcriptomes are besides the direct result
of the DNA sequence, also affected by the epigenome and epigenetic mechanisms, and recent studies suggest a
possible role for epigenetics in the molecular basis of heterosis. In the present report we address the role of
epigenetics in hybrid vigor, and aim to provide a clear and thorough overview of the recent findings on epigenetic
dynamics in hybrids and allopolyploids, the mechanisms involved, and their potential contribution to heterosis.
The reviewed research demonstrates the occurrence of many changes in the state of epigenetic components in
hybrids and allopolyploids relative to the parents at the genome-wide level, as well as at a locus and site specific
scale, allowing potentially for many situations with altered gene regulation that might contribute to heterosis.
Several findings indicate that the alteration of circadian rhythms and expression of downstream genes, resulting
from changed epigenetic patterns may constitute an important and direct way by which the epigenome might
contribute to heterosis. Alterations in the state of epigenetic components occurs mostly at sites with differential
epigenetic states in the parents, and appear to be associated with the presence of siRNAs, that are suggested to
direct methylation changes through the RdDM pathway. Moreover, siRNAs likely play a further role in maintaining
genome stability of hybrids and allopolyploids. More research remains necessary in order to fully understand the
molecular basis of heterosis, which might eventually help us to select the best parent combinations for hybrid
production, specifically manipulate genetic and epigenetic components in order to improve plant characteristics,
and provide food and other materials for the growing human population.

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Introduction
Heterosis or hybrid vigor
Heterosis or hybrid vigor is the phenomenon in which hybrid progeny originating from a cross of two parents that
belong to either different ecotypes, varieties, or cultivars of a single species, or belong to different (sub-)species,
possess trait characteristics that exceed those exhibited by either parent [12, 52, 53]. Although heterosis strictly
covers both positive and negative exceedances, most often the term is used in situations where the trait value of
the progeny has increased compared to the parental state. Heterosis thus refers to trait-speciﬁc measurable
phenotypes and often is related with vigor, which might be plant size, biomass, growth rate, (seed) yield, and
stress tolerance; nevertheless, the term heterosis can be applied to any trait in which the hybrid offspring
surpasses the parents [12, 25, 52, 53, 55]. Nonetheless, people frequently refer to certain hybrids as being less or
more heterotic than others. This description of hybrids as a whole as having low or high levels of heterosis is
based on the false assumption that heterosis is an organism wide phenomenon that involves all traits [25];
heterosis however, as stated previously, is trait specific. Inbreeding depression is regarded as being the contrary
of heterosis, as inbreeding in a population causes the aggregation of detrimental characteristics such as reduced
fertility, lower disease resistance, and slow growth [11]. It is generally assumed that the degree of heterosis
displayed by specific traits of hybrid progeny, correlates positively with the genetic distance between the parents,
suggesting that heterosis requires variation, which might be of genetic or perhaps of epigenetic origin [3, 52, 53,
55]. In support of this, it has been reported for rice, maize, A. thaliana and tobacco that hybrids originating from a
cross of intraspecific parents, which are believed to exhibit relatively little genetic variation compared to
interspecific parental lines, indeed do display heterosis [31, 53], but that the hybrids of inter(sub)specific crosses
show 8-15 % higher values of heterosis for particular traits (in rice) [55, 66-68]. Apart from the variation between
the parents, the level of heterosis observed can also be affected by the stage in development, the environment,
reproductive mode, species type, and varies depending on the trait studied [52, 55].
The phenomenon of hybrid vigor is not restricted to diploid hybrids, and is also exhibited by polyploid plants,
which contain more than two sets of chromosomes [11]. Polyploids display heterosis relative to their diploid
ancestors; moreover, they also display a special form of heterosis known as progressive heterosis, and its study
has been regarded by some to be of major importance for the understanding of heterosis in general [12, 55, 72].
Progressive heterosis occurs when crossing different polyploids, and is believed to be caused by the increased
genetic diversity obtained in the offspring relative to the parents [12]. The classification of polyploid plants
distinguishes two groups, which includes the autopolyploids, resulting from an intraspecific cross (or union of
non-reduced gametes) followed by genome doubling, and the allopolyploids, resulting from interspecific
hybridisation followed by genome doubling [2, 12, 55, 75, 76]. As with inter- and intraspecific hybrids,
allopolyploids exhibit higher levels of heterosis than autopolyploids, which is assumed to be due to the larger
genetic distance between the parental lines [11]. Another factor that has been reported to be a determinant of
the level of heterosis in polyploids is the degree of ploidy [12, 77]. The level of heterosis has been reported to
correlate positively with increasing ploidy. This has been observed in diploid to tetraploid to hexaploid wheat, in
diploid to tetraploid maize (Zea mays) and alfalfa (Medicago sativa), as well as in the octoploid triticale, the
allopolyploid obtained from wheat and rye (Secale cereal) [11, 12, 77-80]. Proposed explanations for this effect
are an increased allelic heterozygosity, and increased gene expression levels resulting from a higher genome
dosage [11]. In contrast to autopolyploids, allopolyploids have been reported to obtain genetic stability over time,
and allow for the permanent fixation of hybrid vigor (through selfing) relative to the original parental lines [2, 10,
55, 84]. It is therefore not surprising that many crops such as cotton (Gossypium), wheat (Triticum aestivum) and
canola are allopolyploid plants. [2, 11, 55].

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Importance of hybrid vigor
At present the use of hybrid crop plants in agriculture is widespread. This is mainly attributable to the increased
yield and uniformity of hybrid plants compared to inbred lines or open-pollinated plants, as well as to their
profitability [3]. Profitability is an important aspect, since farmers have to acquire fresh hybrid seeds every year,
because the full extent of heterosis is limited to the F1 generation, and farmers will only do so when the profit of
the increased yield greatly exceeds the higher yearly costs [3].
As described in Box 1, the study and use of hybrid crop species in agriculture commenced with hybrid maize in the
early 20th
century. Since that time a variety of other crop species has been produced and grown as hybrids. These
include: sorghum (S. bicolor), eggplant (S. melongena), tomato (S. lycopersicum), onion (A. cepa), chili pepper
(Capsicum), rice (O. sativa), peanut (A. hypogaea), cotton (G. hirsutum), wheat (T. aestivum), canola (Brassica),
sunflower (H. annuus), beet (B. vulgaris), and rapeseed (B. napus) [3, 23, 55, 87]. For many crops the production
of hybrid varieties has vastly increased their yield and usage, resulting in a reduced implementation and
popularity of inbred or open-pollinated lines by farmers. Maize yields for example, have increased more than
eight fold since hybrids were first used in the 1930s, to 8 tons per acre [9, 11]. Rice, the staple cereal in China,
India, and other Asian countries, is also grown as hybrids on the majority (55 %) of all rice covered surfaces,
resulting in an increase in yield of 20 to 30 % compared to the best inbred rice cultivars [55, 88]. Also wheat, the
world’s most important cereal crop, is predominantly grown as a hybrid, which exhibits a 10–25 % increase in
agricultural important traits relative to inbred lines [55]. Sorghum, which displays a yield increase of 35–40 % in
hybrid varieties, was globally cultivated as a hybrid on almost half of all plantations at the end of the 20th
century
[55]. Moreover, about 70 % of the 16.5 million hectares planted with sunflower, is covered with hybrids [55]. It is
clear that hybrid plants displaying heterosis are of great importance in providing ample food for the human
population. However, despite their long and widespread use, the precise mechanisms by which heterosis arises
remain largely unknown.
Box 1: The beginning of plant heterosis research and its application
One of the earliest publications on the occurrence of hybrid vigor dates back to 1876, when Charles Darwin published a book with the title “The Effects
of Cross and Self Fertilization in the Vegetable Kingdom”, in which he described examples of experiments he had performed [8-10]. In one of the
experiments, the characteristics of inbred and cross-pollinated (hybrid) maize were compared. Darwin observed that the offspring of the cross-
pollinated maize had an increased overall height of 25 % compared to the selfed inbreds, and noted that the cross-pollinated plants displayed an
elevated tolerance to cold. Overall, Darwin’s book could be summarised by the notion that cross-fertilisation is beneficial, while inbreeding normally has
detrimental outcomes. His findings prompted William Beal to perform hybridisation experiments with open-pollinated (genetically diverse) maize
varieties. Like Darwin, he observed that hybrids displayed increased vigor, and perhaps more importantly showed an increased yield of grain [9].
However, although Beal had demonstrated a potential way to increase the yield of maize plants, the significance of this research was not noticed by
others [9, 27]. In 1908, G.H. Shull published the influential paper “The composition of a field of maize”, in which he demonstrated that the hybrid of two
different inbred lines “is thus immediately brought into harmony” in yield and vigor, with respect to the detrimental effect of inbreeding on both traits
[8, 9, 31]. Moreover, the obtained hybrid progeny was highly uniform in appearance [8, 9]. Around the same time, a scientist named E.M. East
performed similar experiments, but although East noticed the effect of inbreeding, he failed to see the importance of creating hybrids from inbred lines.
In 1909, Shull published a paper describing a procedure for the large scale production of maize hybrids from inbred lines to increase yield compared to
the widely used open pollinated lines. This procedure became the standard in later maize-breeding programs [8, 9]. Although at that time the
phenomenon of hybrid vigor had been described and recognised, the term ‘heterosis’ did not exist yet. It was coined by Shull in 1914 to replace the
word ‘heterozygosis’, and describe hybrid vigor [8, 9, 32]. The word ‘heterosis’ was at the time (and at present) used as a synonym for ‘hybrid vigor’,
Shull preferred the term ‘heterosis’ as it a had more extensive coverage, not only being applicable to an increase in vigor [32]. In the early days of hybrid
maize use in agriculture, the inbred lines used to obtain hybrid seeds were of poor quality and produced too little seeds to be profitable [8, 9, 46]. The
proposition to use double-cross hybrids (cross of two different hybrids) circumvented this issue, and enabled the large scale production of hybrid seeds
[8, 9, 46]. During the following years farmers rapidly made the step from open-pollinated varieties to double cross hybrids; for example, in Iowa their
use increased from 10 % to 90 % between 1935 to 1939. On a national wide scale, the majority of the maize farmed in the U.S. was hybrid by the 1950s
[8, 9]. By the 1960s inbred lines had reached a sufficient quality to be used in the production of single cross hybrids, which were more uniform than
double cross hybrids, and higher yielding [8, 9, 27]. From that time, the application of hybrid maize and other plant species continued to increase, up to
the present.

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Scope of the report
The phenomenon of heterosis, especially in diploid hybrids, has been recognized since 1876, and has been
applied in agriculture for over hundred years [8, 9]. Plants displaying heterosis have allowed us to drastically
increase the yield for various crop species, thereby serving as a food source for many. Nevertheless, despite of
many decades of research directed at unravelling the mechanisms underlying heterosis, our understanding of the
subject is far from complete. However, as it was evident early on that genetic variation between parental lines
often determines the extend of hybrid vigor, early (and later) studies aimed to provide models, and described
heterosis using classical genetics [52, 72, 89-95]. Each of these models has been demonstrated to be suitable for
explaining certain situations in which heterosis occurs [93, 95], but all have limitations and are unable to serve as
a universal explanation for the occurrence of heterosis on their own [25, 90, 92]. Although the degree of heterosis
has been suggested to correlate positively with the genetic distance between the parents, high levels of heterosis
have been observed in hybrids from parents with a highly similar genetic backgrounds, such as closely related
Arabidopsis ecotypes [96]. The epigenomes of these ecotypes do differ, suggesting that existence of epigenomic
variation might have been sufficient for the occurrence of hybrid vigor. Moreover, in recent years, the
phenomenon of hybrid vigor has also been investigated from the viewpoint of epigenetics, and although the
study of the role of epigenetics in heterosis is fairly young, a number of studies has observed that certain aspects
of the epigenome are altered in hybrids and allopolyploids displaying heterosis compared to the parental
situation [5, 10, 45, 53, 97-104]. These alterations in the epigenetic states of hybrids and allopolyploids can
potentially result in changed expression patterns and might lead to an altered transcriptome. This suggests that
heterosis might, besides a genetic basis have an epigenetic basis. However, how epigenetics and possible changes
in the epigenomes of hybrids and allopolyploids might precisely contribute to heterosis is far from completely
understood.
In the present report we focus on the possible role of epigenetics in hybrid vigor, and aim to provide a clear and
thorough overview of the recent findings on epigenetic dynamics and alterations in hybrids and their potential
contribution to heterosis. Nonetheless, we first present the main (historic) genetic models and hypotheses on
heterosis, as well as their limitations in order to provide a basic understanding on the role of genetics in heterosis,
before proceeding to the main subject of this report. In the subsequent sections, we review the current state of
research on the role of epigenetics in hybrid vigor. We present the latest findings regarding the inheritance of
epigenetic modifications and small RNAs in hybrids and allopolyploids, and changes that occur in epigenetic
patterns compared to the parental state. Moreover, we provide evidence of, and speculate how these epigenetic
changes may potentially contribute to hybrid vigor. Additionally, we describe the current models explaining the
observed epigenetic dynamics of heterotic plants, touch upon other phenomena with similar epigenetic
dynamics, and speculate on role of epigenetics in the restriction of maximum heterosis to the F1. Further, the
epigenetic dynamics at fertilisation and the involvement of epigenetics in hybrid incompatibility and parent of
origin effects are addressed. Lastly the general difficulties in studying the role of epigenetics in heterosis are
discussed. We conclude with a recapitulation of the main findings, questions that remain to be answered, and
present suggestions for future research.

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The genetic basis of heterosis
Genetic models
Since the first studies on heterosis, models have been proposed to explain the possible genetic mechanisms
responsible for the observed phenotypes of F1 hybrids displaying hybrid vigor, and the clear transcriptome
changes that occur relative to the parental plants and are responsible for the phenotype [3]. The first of these
models were the dominance model, and the overdominance model [8, 12]. Later pseudo-overdominance and
epistasis were introduced to explain heterosis, and more recently the gene balance hypothesis, and selective
protein synthesis and metabolism hypothesis have been proposed. Here we will briefly describe the genetic
models and hypotheses on heterosis, as well as their shortcomings.
Dominance
The dominance model was one of the first models used to explain heterosis. It was introduced by Keeble and
Bruce in 1910, and elaborated by Jones in 1917 [91, 105]. According to East, 1936: “The explanation of heterosis
(by the dominance model) was so probable that it was generally accepted in spite of the fact that there is no
direct proof for it”. Nevertheless, the model was very popular as it was able to explain the “phenotypic recovery”
of inbred lines suffering from inbreeding depression, when they were crossed to obtain an F1 hybrid [8, 90, 105].
The use of the dominance model decreased from the 1940s when other models gained in popularity [12, 72].
According to the dominance model, which is also known as the complementation model, the increased vigor
observed in hybrids of the first filial generation results from the complementation of (slightly) deleterious
recessive alleles present in the genome of one inbred parent by dominant or partially dominant favourable alleles
from the other parent (figure 1a), following a cross of divergent parents [8, 25, 91, 106, 107]. In order for
complementation to take place, the genomes of parental plants need to contain many deleterious recessive
alleles at different loci [32]. Taken to the extreme, it is possible that the genome of a parent contains genes that
are absent in the genome of the other parent used in the cross. The result is that the genome of the F1 hybrid
comprises an increased number of favourable genes relatives to either of its parents [12]. It has been proposed
that according to the dominance model it should be possible to obtain a homozygous “inbred” from hybrid
parents that contain the maximal amount of favourable alleles, therefore displaying the same amount of vigor as
its parent [25]. This requires sufficient knowledge of recombination events that have to take place, as well as the
occurrence of these recombination events itself, and stringent selection for the right combination of alleles in the
parental hybrid. With the many genes plant genomes contain it does not seem likely that the production of such
an “inbred” is practically feasible.
Seen through the dominance model hybrid vigor is frequently regarded as the result of the maximization of
heterozygosity [25, 106]. Heterosis in that sense is the opposite of inbreeding depression, which is the is the loss
of vigor, fitness or fertility due to mating with genetically related individuals (inbreeding) that results in the
sequestration of homozygous deleterious alleles (reduction in heterozygosity) in the plant genome. Under the
dominance model, both heterosis and inbreeding depression thus have in common that individuals with increased
homozygosity (inbred parents and inbred progeny respectively) generally display reduced fitness [25, 106, 108].
The dominance model also has limitations and not surprisingly therefore has been criticized [12, 25, 109].
Schnable et al. (2013) for example argues that the complementation of detrimental recessive alleles in the hybrid
is not likely to occur in crop species as the genomes of parental inbreds have been under stringent selection for
agronomic traits [25], and this stringent selection is believed to have removed detrimental recessive alleles.
However, stringent selection by plant breeders is probably applied to a limited number of agronomic traits and
underlying alleles of a crop species line. Other traits (and alleles) which are not under selection might in that
situation be allowed to deteriorate, and thereby detrimental recessive alleles may still accumulate. Others have

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also criticized the assumption of the dominance model that homozygous detrimental recessive alleles are widely
present in plant populations [12]. In healthy populations with ample dominant favourable alleles this would
contradict with a principle of population genetics, which states the genotype that is most reproductively fit
dominates. The main drawback of the dominance model however, is that although the complementation of
recessive alleles does take place in hybrids, and is able to explain heterosis for specific loci and traits, it is not
explicable to all situations as studies have shown other mechanisms are often involved [52, 106, 110, 111].
Figure 1: Genetic models for heterosis. (A) The dominance model. Complementation of deleterious recessive alleles a, b, and c by functional dominant
alleles A, B, and C on the other chromosome of the F1 hybrid, and the presence of additional gene D results in heterosis. (B) The overdominance model. The
presence of allele variants C1 and C2 in a heterozygous state in the F1 hybrid causes heterozygote superiority that results in heterosis. (C) The pseudo-
overdominance model. The repulsion-phase linkage of genes C and D in the opposite form of the hybrids chromosomes results in the complementation of
deleterious recessive alleles c and d, by the functional dominant alleles C and D, which mimics over-dominance and causes heterosis. (D) Epistasis. The
combination of alleles A+ and C+ in the F1 hybrid, which can perform inter-allelic interactions, has a multiplicative effect on a vigor related trait and results in
heterosis.
Over-dominance
Together with the dominance model, the over-dominance model was one of the first attempts to explain
heterosis. Shull and East (1908) were early advocators of the over-dominance model, although at the time no
evidence nor any molecular understanding of how it could cause heterosis was available [8, 31]. Mechanisms of
how the over-dominance model could explain heterosis were presented by Hull in 1945 [92], but the over-
dominance model only strongly gained in popularity in 1952 [8].
The over-dominance model (Fig. 1b) states that hybrid vigor arises from heterozygote superiority at genetic loci,
and the increase in vigor correlates with the level of genome-wide heterozygosity [8, 25, 90, 107]. The presence
of different alleles for a single genetic locus results in a better performance relative to that of either allele when

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being present as a homozygote. Over-dominance is reported to be caused by two different mechanisms. It can
result from intra-allelic interactions between the different available allele variants for a specific genetic locus, or it
can be caused by dosage-dependent expression that results in favourable level of gene product, thereby causing
the over-dominant phenotype [106]. In contrast to the dominance model, it is not possible according to the over-
dominance model to obtain from a hybrid an inbred with a level of vigor that is equal to that of the parental
hybrid [25].
Already in the early days of the study of heterosis, the over-dominance model was questioned since there was
limited proof [112-114]. Within a few years after the strong increase in the models popularity, people began to
question the over-dominance model, and although several examples have been described in which over-
dominance has been demonstrated, such as for the SFT gene of tomato which drives heterosis for yield, the
erecta locus in A. thaliana [52, 93, 110], on many occasions what had appeared to be over-dominance in reality
was due to other mechanisms such as pseudo over-dominance [8, 115].
Pseudo overdominance
The mechanism behind the pseudo-overdominance
model was proposed by Crow in 1952 as an
alternative to over-dominance [115]. Pseudo-
overdominance (Fig. 1c) is in reality a version of the
dominance model, and actually serves to explain
situations were over-dominance appears to occur,
instead of being a model that explains heterosis [12,
25, 115]. Pseudo over-dominance involves
complementation of recessive deleterious alleles by
dominant functional alleles like under the dominance
model. Pseudo over-dominance can occur when
parental inbreds with homozygous allele pairs,
possess genomic regions where two or more genetic
loci show little or no recombination events due to
their close vicinity to each other, and are therefore linked. Linked genes in repulsion-phase linkage (Fig. 2), which
means dominant positive alleles are linked with recessive deleterious alleles, can result in multiple
complementation events in the hybrid when the genome of the other inbred parent caries these linked genes in
opposite repulsion-phase linkage. Due to the close linkage of the genes it appears that over-dominance is
operating when not studying the region in detail. Homozygote inbred parents display reduced vigor relative to the
heterozygote hybrid as they possess recessive deleterious alleles which are complemented in their offspring
[108]. Indeed, it has been shown that in several situations where over-dominance appeared to occur, a locus was
composed of multiple QTLs in repulsion-phase linkage [106, 115, 116].
Epistasis
The third model proposed to explain hybrid vigor was epistasis [117]. Epistasis was first discussed by Powers in
1944 as a possible explanation for heterosis [95], but it received little attention [107, 118], and it lasted until 1985
when Willham and Pollak finally described quantitative heterosis theory for two-locus epistasis.
Epistasis (Fig. 1d) results from inter-allelic interactions between several genetic loci. Individual genes themselves
may not contribute to an observed heterotic phenotype. However, when several genes each of which can affect a
shared trait only in combination with specific other genes, is present in a genome with the right genetic
combination, they can together have a multiplicative effect on a trait [106, 108]. This can result in an increase in
vigor of the hybrid when the inbred parents, which both possess genes with allele variants that are unable to
Figure 2: Repulsion-phase linkage of dominant alleles. Alleles A and B are in
repulsion-phase linkage on the genome of both inbred parent P1 and P2.
Hybridization results in the complementation of recessive deleterious alleles a
and b, by the dominant alleles A and b on the opposing strand.

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perform inter-allelic interactions and thereby cause a multiplicative effect on a trait, cross to form a hybrid with
the correct allele combination where inter-allelic interactions do take place, and therefore can strongly affect a
trait.
Early studies on maize grain yield reported that epistasis did not play an important role [107]. Although epistasis
has not been regarded to be as important in heterosis as other models, it has been shown to be applicable to
certain hybrids [61]. Epistasis has for example been reported to play a role in heterosis of certain rice hybrids
[106, 119], many growth variables of Cassava [111], and in heterosis for biomass-related traits in A. thaliana
[120].
Gene balance hypothesis
The gene balance hypothesis as an explanation for heterosis is relatively new [121]. The gene balance hypothesis
was described in great detail and extended by Birchler and Veitia in 2007. Its main principle is that it is of critical
importance for an organism to achieve and maintain favourable balances on a cellular level for components that
are dosage sensitive, such as proteins with opposing actions or protein-complex subunits [25, 121]. Optimal
balances should be maintained as deviations from the equilibrium state might negatively affect an individual’s
fitness. The gene balance hypothesis argues that due to the heterozygous state of many genes in a hybrid
obtained from inbred parents, hybrids will have more dosage sensitive components that display an optimal
balance relative to its parents. Especially when a trait is affected by several genes, the heterozygous state of allele
pairs allows the hybrid to modulate the phenotype of the trait as a result of the different alleles available by
which balances can be optimised. Therefore, hybrids will show heterosis for certain traits compared to their
parents. The gene balance hypothesis was not intended as a better or alternative explanation for heterosis than
the dominance model, over-dominance, or epistasis, but rather as an additional hypothesis for situations where
the other models could not provide adequate explanations.
Selective protein synthesis and metabolism hypothesis
The most recent model for explaining heterosis is the selective protein synthesis and metabolism hypothesis,
which was advanced by Goff in 2011 [117]. The central message is the increase in energy efficiency of hybrids due
to selective synthesis of protein and limited protein metabolism, which results in increased vigor. Important in
this hypothesis is that allelic variants often encode inefﬁcient, improperly folding, and above all unstable and
therefore unfavourable proteins [25, 106, 117]. Inbreds therefore have a high metabolic level of unfavourable
proteins, as they have many homozygous deleterious allele variants. The high metabolism is due to the absence
of the possibility to select from different, more favourable alleles. As a result of the high protein metabolism,
which is energy intensive, little energy is left for the inbred plant to invest in growth. Hybrids on the other hand,
with many loci in a heterozygous state, might possess more and less favourable alleles for a specific gene. This
allows for the possibility to select the preferred allele for the encoded protein, which decreases the cellular level
of protein metabolism and therefore energetic cost. More energy is available for growth, which shows itself as an
increase in vigor. The selective protein synthesis and metabolism hypothesis is also able to explain heterosis
observed in allopolyploids. Like diploid hybrids, allopolyploids will show hybrid vigor as they have a larger
selection of alleles to choose from, and thus to reduce metabolic costs.
Selective regulation of protein synthesis and metabolism implies an active monitored process, and although Goff
proposed a cell-based quality control mechanism that includes the comparison of the stable and less stable gene
product during translation, degradation of unstable, improperly folding and aggregated protein and its mRNA,
and finally epigenetic silencing of the unfavourable gene, he did not show evidence of the existence of such a
process [117, 122]. It is therefore not likely that such a mechanism is responsible for the positive heterosis often
observed in F1 hybrids.

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Limitations of genetics in explaining heterosis
The genetic explanations for heterosis are based on a limited number of hypotheses: dominance, overdominance,
(pseudo over-dominance), and epistasis which have dominated the literature for the last century [12]. Despite
their popularity, and notwithstanding their ability to explain heterosis in certain situations, neither of these
models on its own is sufficient to explain all instances in which hybrid vigor occurs. Furthermore, studies using
similar rice hybrids have proposed different genetic models for the observed heterosis [112-114]. Although these
studies were performed by different groups and directed at different loci, it highlights the inability of each model
to serve as a universal explanation for heterosis. The main limitation of the older classical genetic models, is that
they are inadequate to explain the molecular basis of hybrid vigor as these models do not involve molecular
principles [11, 24, 88].

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Epigenetics and heterosis
For a long time, genetic theories explaining heterosis as described previously dominated the research on
heterosis. In recent years however, the possible role of epigenetic mechanisms in the molecular basis of heterosis
has attracted a growing amount of attention, and an increasing number of studies on the subject has been
published. Epigenetics is the study of alterations that occur in gene expression and in the regulation of gene
activity, which are not the result of changes in the genetic sequence of cells or the individual organism [123]. The
term epigenetics has often been argued to require a heritable state [123], able to be stably transmitted through
one or more cycles of meiosis or mitosis. In the current report the definition is used as stated by the NIH
Roadmap Epigenomics Mapping Consortium: “epigenetics refers to both heritable changes in gene activity and
expression (in the progeny of cells or of individuals) and also stable, long-term alterations in the transcriptional
potential of a cell that are not necessarily heritable” [124]. This definition includes changes resulting from
epigenetic components as DNA (cytosine) methylation, histone modifications and certain types of small RNAs and
associated mechanisms [125].
DNA methylation is the addition of a methyl group by DNA methyltransferases to the position 5’ of cytosine bases
(5mC) [3, 4, 24]. In contrast to mammals, which are almost exclusively able to methylate the cytosines of CG
motifs, plants are able to methylate cytosines within CG, CHG, or CHH motifs (in which H can be A, T, or C). The
catalysis of 5mC by DNA methyltransferases can take place at cytosine bases that have previously been free of
methylation (de novo methylation), or might occur at cytosines that have lost pre-existing methylation in order to
maintain methylation patterns (maintenance methylation). Maintenance methylation occurs mainly at the
symmetrical sites of CG and CHG motifs. Due to the symmetry of these motifs, methylating enzymes are able to
transfer the methylation pattern present on the mother strand to the daughter strand after DNA replication (Box
3). De novo methylation at all three motifs, and maintenance methylation of CHH sites occurs mainly through the
RNA dependent DNA Methylation pathway (RdDM pathway, described in Box 6), which depends DOMAINS
REARRANGED METHYLTRANSFERASE2 (DRM2), and involves 24-nt short interfering RNAs (siRNAs) that have been
reported to function in guiding the proteins responsible for DNA methylation to their target site [3, 4, 22-24].
Recently however, alternative pathways responsible for de novo methylation have been discovered [1, 2].
Cytosine methylation has been reported to occur primarily in repetitive regions enriched in transposable
elements, silent 5S and 45S rRNA repeats, and centromeric repeats [4, 23, 24, 26, 126, 127], where 5mC is mainly
believed to function as a silencing mechanism that maintains genome stability by repressing repetitive regions.
5mC further occurs at pseudo-genes, intergenic- and intragenic regions, where it is associated with differentially
regulated promoters and transcribed regions of moderately expressed protein-coding genes. The active removal
of methylated cytosines is also involved in biological processes, such as gene imprinting [125]. In A. thaliana, DNA
glycosylases are responsible for the active de-methylation of cytosines, and play roles in several developmental
and physiological processes in plants [4, 24].
Besides cytosine methylation, small RNAs form an important part of the epigenome. Small RNAs (sRNAs) are RNAs
typically 20–30 nucleotides in length, and are important in epigenetic regulation [2, 15]. They have been
identiﬁed as regulators in plants, participating in the regulation of plant growth and development, adaptation to
abiotic stresses and responses to biotic pathogens [1]. Two major groups of small RNAs are distinguished, the
small interfering RNA (siRNA) and micro RNA (miRNA), which are produced by different ways. Short interfering
RNAs (siRNAs) in plants are mainly 21, 22 and 24 nucleotides in length, and are produced directly from
endogenous loci and repetitive regions, or indirectly in response to exogenous agents such as viruses, and their
biogenesis start with transcription by DNA DEPENDENT RNA POLYMERASE IV (Pol IV) (Box 4) [2]. Mature siRNA
incorporated in protein complexes participates in the process of transcriptional gene silencing (TGS), or in post-
transcriptional gene silencing functions (PTGS) through pairing with mRNA sequences complementary to that of
the siRNA followed by mRNA cleavage (Box 4) [1], the latter which is mainly characteristic for 21-nt and 22-nt

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siRNAs [4, 33, 35, 36, 40, 41]. In A. thaliana the majority of siRNAs is 24-nt in length, and is mainly produced from
transposons, repeat sequences, and a limited number of pseudogenes [50, 62]. DNA sequences generating 24-nt
siRNA are often present as siRNA clusters (SRCs), which may contain thousands of siRNA encoding sequences [1,
45, 128, 129]. In contrast to the 21-nt and 22-nt siRNAs that are mainly involved in PTGS, 24-nt siRNAs play roles
in directing epigenetic modifications (DNA methylation) to homologous DNA sequences, as stated previously,
resulting in the transcriptional silencing of genes and mostly TEs, through the process of RNA-directed DNA
methylation (RdDM, Box 6). The micro RNAs constitute a class of small RNAs generally 20–24 nucleotides in
length, with the most of them being 21-nt; in A. thaliana 80 % of the miRNAs is 21-nt long, which is more than in
rice and maize [2, 15]. MiRNAs play many important roles in plant developmental processes such as stem cell
maintenance and differentiation, organ specification, elongation, flowering and fertility, etc. [1, 4]. Micro RNAs-
are able to cause PTGS due to mRNA slicing by a protein complex or by inhibiting translation due to the
complementarity of mRNA to the miRNA [2, 73, 74]. In plants miRNAs can also initiate the production of
secondary 21-nt long trans acting siRNAs (ta-siRNAs) [83], which are trans acting because they target loci that are
distinct from the TAS loci from which they originate. They can induce PTGS, much like miRNAs [1, 2, 4, 15, 83].
However, similar to certain siRNAs, some ta-siRNAs and miRNAs play roles in TGS through alternative RdDM
pathways (Box 6).
Apart from the methylation of DNA, chromatin can undergo several modifications to the histones that form the
nucleosome, which constitutes the most elementary packaging form of chromatin, and is composed of histone
variants H2A, H2B, H3 and H4 that arrange to form an octamer around which DNA with a length of 147 bp is
wrapped [23]. Histone modifications can affect gene expression activity in response to diverse endogenous and
exogenous stimuli, by affecting the accessibility of proteins such as transcription activators and repressors, or RNA
polymerases to specific target sequences in the DNA [23, 125]. Histone modifications are covalent modifications
at the N-terminal tail of histone proteins, which are reversible and dynamic [125]. Histone acetylation and
methylation at lysine residues are two of the most studied epigenetic marks, and are established by histone
acetyltransferases (HATs) and histone lysine methyltransferases (HKMTs) respectively (Box 5). The addition of
histone modifications may have different effects on chromatin structure and expression activity of genes (Box 5),
such as a more open chromatin state and consequently gene activation, or a more condensed chromatin state
and thereby gene repression. Acetylation of lysine residues at histone H3 is typically a characteristic modification
of active protein coding genes [13, 39]. The effect of lysine methylation of histone H3 on the other hand depends
on the degree of methylation, as well as the position of the methylated lysine residue in the N-terminal tail [24,
130], and can be associated with active gene expression or repression.
Epigenetic components such as DNA methylation, small RNAs and histone modifications are heritable, but also
dynamic and reversible, and diverge quickly [1, 23, 45, 96]. This dynamic nature may serve as a fast and flexibly
way to adjust the transcriptome in response to varying environmental conditions, in order for plants to survive
and thrive, but also causes closely related species to acquire different epigenomic compositions. Moreover, due
to the dynamic nature of epigenetics, it is possible that the parental state of epigenomic components becomes
differentially inherited in the progeny, which can result in the formation of new patterns in hybrids and
allopolyploids that did not exist in the parents. This might result in altered gene regulation and expression, and
may cause changes to the transcriptome, which could thereby possibly contribute to heterosis.

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Effects of hybridization and allopolyploidization on genome-wide epigenetic and small RNA patterns
of heterotic progeny
In recent years several studies have investigated the genome-wide patterns of small RNAs, cytosine methylation
and histone modifications in heterotic hybrids, allopolyploids, and their parents, as well as their relationship with
genetic variations and phenotypic diversity. This in order to determine whether hybridisation and
allopolyploidization results in epigenomic changes that might contribute to hybrid vigor. Although often mostly
additive inheritance (term explained in Box 1) of epigenetic components has been observed in hybrids and
allopolyploids in these studies, many situations with non-additive inheritance have also been detected, which
may potentially result in altered gene transcription.
DNA methylation Regarding the genome-wide level of DNA methylation, iIn intra-specific hybrids of rice NPB and
93-11 strains, the genome-wide level of cytosine methylation was found to be mostly similar to the parental state,
as only 0.8 % of the cytosines was differentially methylated when accounting for cis genetic differences [99]. Also
in intra-specific Arabidopsis hybrids of several ecotypes (C24 x Ler, Col x C24, Col x Cvi, Ler x Cvi), the global level
of cytosine methylation was found to be similar to that of the parents [98, 131, 132]. This suggests that additive
inheritance of methylation levels is the main pattern of inheritance for DNA methylation in Arabidopsis (~3 % non-
additively methylated) and rice intraspecific hybrids [98, 99, 131, 132]. However, in other studies the genome-
wide methylation level has been observed to be increased in intraspecific Arabidopsis C24 x Ler and Populus
hybrids compared to the midparent value (MPV, elaborated upon in Box 1) [103, 133, 134]. Nonetheless, in
Arabidopsis the percentage of CG, CHG and CHH (together denoted as C/G) sites or clusters, with similar or
additive methylation levels as the parents has been reported in several articles to be over 90 % [98, 131, 132,
Box 2: Descriptions of phenotypic levels in hybrids and allopolyploids
The phenotypic level of any (molecular) trait in a hybrid or allopolyploid can be described with several terms. The hybrid phenotype of traits such as
methylation level or gene expression can for example be described as being additive compared with that of the parents. In the event that the
methylation level or gene expression is additive (Fig. 3), this means that it is equivalent to (not significantly different from) the midparent value (MPV)
[11, 12]. Molecular traits and large scale traits related to vigor that display additive or close to additive phenotypic levels, are often considered to be of
limited importance to the displayed heterotic phenotypes, and agronomic use respectively [25]. If the level of a trait is non-additive (Fig. 3), the trait
value of the hybrid is either higher or lower than the MPV. Non-additive patterns can be further classified as having a phenotypical trait level equal or
close to (better-parent heterosis = BP), or greater than (above better parent heterosis = ABP) the level of the parent with the highest value for the
trait; or adversely, as having a phenotypical level that is equal or close to (low parent heterosis = LP), or lower than (below low parent heterosis = BLP)
the level of the parent with the lowest value for the trait [11, 12, 25]. Finally, there is a category of hybrid trait levels that significantly deviate from the
MPV that are classified as below high parent, or above low parent. As stated, the level of a molecular trait in a hybrid or allopolyploid can be additive.
Being non-significantly different from the MPV, additive traits are therefore sometimes considered as being unchanged and un-influenced by the
hybridisation or allopolyploidization event, thereby deserving less attention. However, an additive trait value does not automatically imply that the
allele specific phenotype of the (molecular) trait has not changed. Molecular traits such as the level of cytosine methylation, histone modifications,
and siRNA expression may show differential values between the parental state and the parental allele of the hybrid progeny, while reaching an
additive level for both alleles [11].
Figure 3: Possible phenotypical levels of (molecular) traits in
hybrids or allopolyploids compared to the parental state. The
phenotypical level of molecular traits in hybrids and
allopolyploids can be additive (left column) or non-additive
(middle and right column). An additive trait level indicates that
the average (allelic) phenotypic value of the (molecular) trait in
the hybrid or allopolyploid is equal to the parent average or
midparent value (MPV). A non-additive trait level indicates that
the average (allelic) value of the (molecular) trait in the hybrid
or allopolyploid is statistically significantly different from the
MPV. In this situation the allelic average can be non-additively
decreased or increased. P1 and P2 represent parent one and
two respectively. MPV represents the midparent value. F1
represents both F1 hybrid and allopolyploid progeny. The
figure is adapted from Chen et al. (2013), and adjusted.

- 14 -
134]. With respect to specific methylatable cytosine motifs, Shen, et al. (2012) and Greaves et al. (2014) observed
in the Arabidopsis C24 x Ler hybrid an increase in the genome-wide CG methylation levels of 19 % and 23.2 %
respectively. Nevertheless, as reported above for C/G sites in general, most of the CG motifs showed an additive
pattern of methylation in Arabidopsis hybrids, as only 5 % of the CG sites in clusters was non-additively
methylated [134]. Differing from the previous, Chen et al. (2014) found that global CG methylation of Arabidopsis
hybrids was unchanged compared to the MPV. Global CHG methylation levels have been reported to be increased
(by 26.67 %) [103, 133, 134], or unchanged (C24 x Ler) in hybrids from Populus and Arabidopsis (C24 x Ler) [98].
Notwithstanding this, the inheritance pattern of CHG methylation was found to be mostly additive in Arabidopsis
(C24 x Ler) hybrids, with only 8 % of the CHG motifs displaying non-additive methylation [134]. As for CHG motifs,
CHH methylation at the genome-wide scale of Arabidopsis (C24 x Ler) and Populus hybrids has in several studies
been found to be increased (by 25 % [134]) compared to the MPV [103, 133, 134], and non-additive in ˜22 % of
the mC clusters, which is a larger percentage than for the two other motifs [134]. Another study on the other
hand, observed no change relative to the parents, in the genome-wide CHH methylation level of Arabidopsis (C24
x Ler) hybrids [98].
In interspecific hybrids and allopolyploids, MSAP measurements have shown different inheritance patterns than
found in intraspecific hybrids. In the interspecific hybrid of Chrysanthemum morifolium and Leucanthemum
paludosum for example, the genome wide level of cytosine methylation (~45 %) as measured by MASP analysis
was reduced compared to the parental state (50.6 – 51.5 %) [135]. Similarly the percentage of methylated MSAP
sites was found to be reduced compared to the parents in the interspecific hybrid of B. rapa and B. nigra [136].
Focussing on the different cytosine motifs, an increase was observed in the percentage of methylated CG sites,
which surpassed the high parent value. On the other hand, the percentage of CHG methylation displayed a
reduction to below low parent levels. This suggests that the methylation dynamics at the CHG motif might be
responsible for the observed reduction of genome-wide methylation in the hybrid. In the interspecific hybrid of
Raphanus sativus and Brassica alboglabra, most (7/12) of the tested mC sites were reported to display no change
in their methylation status compared to the parental situation, [137]. Similarly, the percentage of methylated
MSAP sites in the S2 allotetraploid of B. rapa and B. nigra, was reported to be reduced compared to the state in
the parents, and was even lower than the percentage in the hybrid [136]. Both CG and CHG methylation levels at
the tested MSAP sites were lower in the allotetraploid than in the parents, which is different from the hybrid.
Based on these findings genome-wide methylation levels of interspecific hybrids and allopolyploids appear to
mostly reduced compared to the parental situation, in contrast to intraspecific hybrids which display methylation
levels similar to or increased relative to the parents. However, the MSAP technique is not very sensitive, and the
relatively small number of tested mC sites in interspecific hybrids and allopolyploids might have resulted in an
inaccurate representation of the actual situation, and could explain the differences.
Although some studies have found no alteration in the level of genome-wide methylation, others did observe a
decrease or increase in the global methylation level of hybrids and allopolyploids. The same holds when looking at
the global methylation levels of different C/G motifs, which even when altered, displayed mostly additive site-
specific inheritance. Indicating that additive inheritance is the main pattern of DNA methylation inheritance in
hybrids, and suggesting that only a small fraction of all C/G sites is non-additively methylated.
Non-additively methylated sites have been shown to display increased levels of methylation relative to the MPV
in several intraspecific hybrids such as those obtained from Arabidopsis C24 and Ler ecotypes [103, 134], O. sativa
cv. Nipponbare and cv. 93-11 [101], and P. deltoides cl. 55/65 and cl. 10/17, [133]. This increase might explain the
genome-wide increase in methylation observed of some studies described above. With respect to cytosine
methylation at CG motifs, three studies using intraspecific hybrids of Arabidopsis C24 x Ler, and C24 x Col
ecotypes, universally observed that most of the CG sites with non-additive methylation had increased mC levels
compared to the MPV [103, 132, 134]. Similarly, non-additively methylated CG motifs in the S3 wheat

- 15 -
allohexaploid of T. monococcum and Ae. sharonenis were observed to be increased compared to the MPV [138].
This suggest that increased methylation levels at non-additively methylated C/G sites and more specifically CG
motifs is a common property of hybrids and allopolyploids. The genome wide level of CHG and CHH methylation
has been reported to be increased compared to both parents, in the hybrid of Arabidopsis C24 x Ler as stated
above [103, 134]; nevertheless, most (~63.8 % and ~81.3 %) of the CHG and CHH clusters displaying non-additive
methylation showed a decrease in the level of methylation compared to the MPV [134], suggesting that the level
of a small fraction of these clusters with non-additive methylation is strongly increased.
Small RNAs With respect to the genome-wide inheritance patterns of small RNAs in hybrids, the distribution of
small RNA populations over the genome of Arabidopsis C24 x Ler hybrids was found to be unchanged compared
to the parents [53], and in intraspecific hybrids of both monocot maize (B73 x Mo17) and dicot Arabidopsis (Col x
Ler), the small RNA length profile has been shown to be similar to the parents, and small RNAs were mostly
additively expressed [97, 139]. Moreover, no small RNA clusters were identified in Arabidopsis Col x Ler hybrids
that were not expressed in one or the other parent [139], and almost all the clusters (96–99 %) showed a hybrid
level within the parental range. Short RNAs as a whole thus appear to be mostly inherited unchanged.
A number of studies has focussed specifically on siRNAs, which mainly include small RNAs of 21, 22 and 24
nucleotides in length and which are involved in PTGS and TGS (Box 4). In the NPB x 93-11 rice hybrid 78.8 % of the
siRNA clusters was additively expressed [101], and predominantly additive transcript levels were also observed
for siRNA clusters in Arabidopsis Ler x C24 hybrids, although the overall number of siRNA clusters was found to be
increased compared to the parents [103]. He et al. (2010) on the other hand, reported that 18-26 nt sRNAs were
suppressed in F1 rice hybrids. However, after a re-examination of their data they concluded that there was no
support for this statement [103]. Various studies have obtained different results regarding 24-nt long siRNAs,
which originate from repetitive sequences, are involved in TGS and linked to DNA methylation (Box 4, 6) [53, 97,
101, 103]. According to at least two reports on Maize and Arabidopsis hybrids, the genome-wide level of 24-nt
siRNAs was decreased compared to the MPV in the hybrid [53, 97]. In contrast, Shen et al. (2012) observed no
clear changes in the 24-nt siRNA level in the (Arabidopsis) hybrid compared to the parents. Moreover, according
to He et al. (2010) the proportion 24-nt siRNAs form of the small RNA total increased in their intraspecific rice
Box 3: DNA methylation in plants
Plants contain several proteins that are able to methylate DNA, most of which are methyltransferases that belong to one of the three families
that are composed of homologues of mammalian DNMT1, DNMT2, and DNMT3 [3, 4], which are involved in de novo and maintenance
methylation. METHYLTRANSFERASE 1 (MET1) is the plant homologue of DNMT1, and considered to be the main contributor to CG maintenance
methylation. In the process of CG maintenance methylation, MET1 acts together with VARIANT IN METHYLATION 1 (VIM1), which has a
preferential affinity for hemi-methylated CG sites, allowing for the selection of methylation targets on the unmethylated DNA strand. MET1 is
also believed to contribute to de novo methylation of CG motifs. The group of enzymes representing the plant homologues of mammalian
DNMT3 is that of the DOMAINS-REARRANGED METHYLTRANSFERASEs (DRMs), which function predominantly as de novo methyltransferases
[4]. DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) is responsible for de novo methylation at CG, CHG and CHH motifs and involved
in the “maintenance” methylation of CHH sites. CHH “maintenance” methylation is based on continuous de novo methylation through the RNA
dependent DNA Methylation pathway (RdDM pathway, described in Box 6), of which the canonical version depends on DRM2 [3, 4, 22-24].
Until recently CHH methylation was believed to be mainly effectuated by the activity of DRM2; however it has been shown that the plant
specific CHROMOMETHYLASE 2 (CMT2) is responsible for a considerable portion of CHH methylation present in the genome of Arabidopsis,
mainly in heterochromatic regions containing transposon elements (TEs) [3, 4, 15, 22-24]. Unlike DRM2, methylation of the CHH motif by CMT2
has been reported to be independent of the RdDM pathway, and likely occurs by CMT2 binding the heterochromatic histone modification
H3K9me2 through its chromodomain. Binding of di-methylated H3K9 has also been reported for CHROMOMETHYLASE 3 (CMT3), and is
subsequently followed by de novo DNA methylation of CHG motifs at the binding site [3, 4, 24].
In A. thaliana, the DNA glycosylases responsible for active de-methylation are REPRESSOR OF SILENCING 1 (ROS1), DEMETER (DME), or
DEMETER-LIKE proteins (DMLs) [4, 24]. DME has been suggested to be required mainly for CG de-methylation, and has been reported to play
an important role in the gametophytes. ROS1 is expressed in a constitutive manner, and is therefore thought to be in part responsible for the
loss of DNA methylation in non-dividing cells. ROS1 possesses the ability to associate with ROS3, which is believed to be necessary for guiding
ROS1 to its sites of action.

- 16 -
hybrids compared to the MPV. Nevertheless, two of the studies that report contrasting findings with respect to
[53, 101] percentage of 24-nt siRNA in intraspecific hybrids relative to the MPV, both report that 24-nt siRNAs
were mostly additively expressed. In allopolyploids, 24-nt siRNAs were found to show a decrease in percentage of
the siRNA total in the S1 wheat allohexaploid and S2 Brassica allotetraploid respectively [136, 140]. However, S3
and S5 allohexaploids of respectively wheat and Brassica have been shown to display a percentage increase
relative to the siRNA total, of 20.81 % in diploid B. rapa, 37.22 % in tetraploid B. carinata, and 35.27 % in the S5
Brassica hexaploid [141, 142]. Although different results have been obtained for 24-nt siRNAs, most studies seem
to suggest that genome-wide 24-nt siRNAs levels are reduced in hybrids and allopolyploids of the first two
generations, in contrast to later generations. Nevertheless, most 24-nt siRNAs appear to be additively inherited.
Non-additively inherited small RNAs represent only a small proportion of the total in Arabidopsis hybrids.
Nonetheless, they can potentially have a large impact as changed small RNA levels may result in alterations in
small RNA dependent regulation. Downregulation of non-additive small RNAs appears to be a general pattern in
hybrids. In the intraspecific hybrid of Arabidopsis Col x Ler, 98 % of the non-additive differentially expressed small
RNA clusters was decreased in their expression compared to the MPV [139]. Consistent with this, ~81 % of the
non-additive siRNA clusters in Arabidopsis C24 x Ler hybrids showed in a reduction in their expression level
relative to the MPV [103], and in the intraspecific hybrid of O. sativa ssp japonica cv Nipponbare and O. sativa ssp
indica cv 93-11, 67 % of the non-additive siRNAs was reduced relative to the MPV [101]. Non-additive siRNAs in
the intraspecific maize hybrid B73 x Mo17 were also biased to below MPV levels, and more specifically, non-
additive 24-nt siRNAs tended to be downregulated with respect to the MPV [97]. A reduction in the expression
level of non-additive 24-nt clusters was also observed by Groszmann et al. (2011) in their Arabidopsis C24 x Ler
hybrid, where 93.6 % displayed expression levels below the MPV [53].
Regarding the inheritance dynamics of miRNAs, known for their roles in PTGS, varying observations have been
made for intraspecific hybrids [88, 143]. According to Fang et al. (2013) most (18 of the 19 studied) miRNAs were
non-additively expressed in intraspecific ZS97 x MH63 rice hybrids, while Zhang et al. (2014) showed that in their
intraspecific PA64s x 93-11 rice hybrids miRNA expression was predominantly additive (~305 of 350 investigated
miRNAs). This difference however, might be due to several causes such as the small group of miRNAs studied in
the paper of Fang et al. (2013), different plant materials studied (seedling leaves by Fang, et al. (2013),
reproductive stage leaves by Zhang et al. (2014), or cross specificity. In maize hybrids, some of the tested miRNAs
in the B73 × Mo17 cross were non-additively expressed according to Barber et al. (2012), suggesting that most
miRNAs displayed additive expression levels. Although most miRNAs are 21-nt in length, several studies that have
investigated epigenetic mechanisms involved in heterosis, report on 21/22-nt long siRNAs without defining them,
or a subset of them, as miRNAs. A selection of 21/22-nt long small RNAs, might therefore include a combination
of miRNAs, ta-siRNAs and siRNAs. The genome-wide level of 20-22-nt long siRNAs was shown to be unchanged in
the (C24 x Ler) intraspecific Arabidopsis hybrid compared to the parents [53]. However, again in contrast with
Groszmann et al. (2011), Shen et al. (2012) observed an overall increase of 21-nt siRNA levels in the intraspecific
(Ler x C24) Arabidopsis hybrids compared to the parents. Due to their different findings Shen et al. (2012) has
criticized the study by Groszmann et al (2012) by stating that: “The total sRNA clusters obtained in that study only
covered 1.6 - 2.08 % of the genome, far below our total sRNA clusters, which covered 47.27 % of the genome”.
This dissimilarity might explain the different results obtained for the levels of 21-nt and 24-nt siRNAs in their
(Arabidopsis) hybrids. Both used tissue of similar age, being 14 and 15 day old seedlings by Groszmann et al.
(2011) and Shen et al. (2012) respectively, which is therefore unlikely to explain the distinct results. In
intraspecific rice hybrids, the relative abundance of 21-nt siRNAs on the siRNA total increased relative to the MPV,
but the great majority of 21-nt siRNA clusters displayed additive expression levels [101]. As has been found in
several intraspecific hybrids, the percentage of miRNAs of the sRNA total was increased in F1 interspecific hybrids
of Brassica rapa and Brassica nigra compared to the parents [136]. Similar results were found in the triploid

- 17 -
interspecific wheat hybrid of the diploid Ae. tauschii and the tetraploid T. turgidum, where the miRNA fraction of
sRNA total increased compared to the diploid parent and the MPV [140]. Also in allopolyploids miRNAs have been
observed to display proportional increases. miRNAs in the S1 allohexaploid of diploid Ae. tauschii and tetraploid T.
turgidum for example, made up an increased percentage (to 44 %) of the sRNA total compared to both parents
(21 % and 38 %) and the triploid hybrid (33 %) [140], although most of the miRNAs in the hybrid were expressed
additively. Similarly, the percentage of miRNAs increased to 39.22 % in the second generation allotetraploid of B.
rapa and B. nigra compared to both parental values (30.9 % and 29.34 % respectively) and the interspecific hybrid
(31.44 %) [136]. In the fifth generation allohexaploid of diploid B. rapa and tetraploid B. carinata however, the
fraction of miRNAs of the sRNA total was observed to be decreased compared to both parents [142]. Despite the
increased proportion of miRNAs on the small RNA total, micro RNAs were found to be mostly additively expressed
in the S3 allohexaploid of diploid T. turgidum and tetraploid Ae. Tauschii (76.3 % additive) [141], and likewise in
the flowers of the S7 allotetraploid of A. thaliana and A. arenosa [144]. In the leaves, only half of the tested
miRNAs was additively expressed in the S7 allotetraploid. Contrary to the pattern of 24-siRNAs, the percentage of
miRNAs on the small RNA total, and thus presumably the number of different miRNAs expressed, appears to be
increased relative to the parental state in hybrids and first generation allopolyploids, which might affect
developmental processes and so influence plant phenotype [2, 4]. The increase in miRNAs might be due to
expression of both parental miRNA libraries in the hybrid and allopolyploid. Nonetheless, in general miRNAs do
display additive genome-wide expression levels.
In contrast to siRNAs, non-additive miRNAs and ta-siRNAs do not seem to display a clear general pattern of
inheritance in hybrids. In Arabidopsis C24 x Ler hybrid for instance, non-additive 21-nt small RNA clusters showed
similar tendencies for reduced or increased expression relative to the MPV [53]. Ghani, et al. (2014) on the other
hand, observed in the interspecific hybrid of B. rapa and B. nigra that non-additive miRNAs in leaf tissue, mostly
showed an increase in their expression level [136]. Even within a single type of hybrid, different patterns have
been found for non-additive miRNAs in different tissues. Non-additive miRNAs and ta-siRNAs in the PA64s x 93-11
rice hybrid showed in flag leaves an equal tendency to be decreased or increased compared to the MPV, while in
panicles most (~66 %) of the tested non-additive miRNAs and ta-siRNAs displayed a decreased expression level
relative to the MPV [143]. Others have described similar results in the rice hybrid SY63 produced from O. sativa
lines ZS97 and MH63, where in leaves more than half (10/18) of the tested non-additive miRNAs was reduced in
its expression compared to the MPV [88], while in culm and root tissue, half (9/18) and more than half (12/19) of
the tested non-additive miRNAs respectively, displayed a decrease relative to the MPV. Consistent with the
previous, maize hybrids also display different patterns for non-additive miRNAs. In B73 x Mo17 maize hybrids,
non-additive miRNAs appeared to be mostly increased in their expression compared to the MPV in shoot tissue,
while they tended to be decreased in the ear [97]. According to Barber et al. (2012), in maize miRNAs account for
16 % of the shoot apex sRNA population but only 2 % of the developing ear sRNA population, and the miRNA
profiles of the two tissues differ dramatically [97]. These differences in miRNA profiles might explain the different
tendencies of non-additive miRNAs in most hybrids, and suggest that the type of plant tissue more strongly
affects miRNA expression patterns than hybridisation itself. Regarding allopolyploids, studies on Brassica,
Arabidopsis, and wheat have found similar results for non-additive miRNAs [136, 141, 142, 144]. Non-additive
miRNAs in S2, S3, S5, and S7 allopolyploids were mostly reduced in their expression compared to the MPV,
suggesting that this decreased expression level of non-additively expressed miRNAs is a universal pattern in
allopolyploids, irrespective of the filial generation.

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Histone modifications Little attention has been directed to the genome-wide inheritance patterns of histone
modifications that possibly affect the chromatin state of hybrids. In one study, the genome-wide level of
H3K4me2 (chromatin compacting) and H3K27me3 (chromatin opening) in intraspecific Col x Cvi Arabidopsis
hybrids was largely unchanged compared to the parents [145]. Similarly, in monocot rice histone modifications at
genes have been reported to be mostly unchanged in the hybrid relative to the MPV [101]. This was shown as
well for the chromatin opening H3K4me3 and H3K27me3 modifications at TEs. This suggest that for histone
modifications additive inheritance is the main pattern of inheritance in hybrids of both monocot and dicot
species. At regions with non-additive H3K27me3 and H3K4me3, the level was mostly found to be elevated with
respect to the MPV in the intraspecific F1 rice hybrid of O. sativa ssp japonica cv Nipponbare and O. sativa ssp
indica cv 93-11 [101]. A similar result has been found for H3K4me3 in the intraspecific F1 hybrid of maize inbred
lines B73 and Mo17 [45], suggesting that histone modifications with contrasting effects (Box 5) on chromatin
density and thereby gene expression are both increased in non-additive situations found in hybrids.
Box 4: Biogenesis and mechanism of action of small RNAs in plants
siRNA The biogenesis of siRNAs starts with the production by DNA DEPENDENT RNA POLYMERASE IV (Pol IV) of single-stranded RNA (ssRNA) that is
subsequently converted into double stranded RNA (dsRNA) [2]. DICER-LIKE (DCL) proteins are responsible for the production of siRNAs from dsRNA, by
cleaving dsRNA into siRNAs of specific lengths depending on the type of DCL protein [1]. The siRNAs are incorporated into specific ARGONAUTE (AGO)
proteins of RNA-INDUCED SILENCING COMPLEX (RISC) depending on siRNA length. The siRNA carrying RISC subsequently participates in the processes of
transcriptional gene silencing (TGS), or in post-transcriptional gene silencing (PTGS) through pairing with mRNA sequences complementary to that of
the siRNA, which is followed by mRNA cleavage. In plants, siRNAs are mainly 21, 22 and 24 nucleotides in length, whose production mainly starts out
with Pol IV and RDR2, and further resembles the process described above. However, different types of siRNAs can be produced by different paralogous
proteins and may perform different functions.
The 21-nt long siRNAs are the smallest type of siRNA with a considerable abundance in plants. Some of these siRNAs are generated from dsRNA
produced by RDR2, and due to cleavage by DLC1 (which mainly generates 21-nt miRNAs) associate with AGO1, and induce PTGS by mRNA slicing [33].
Most 21-nt and 22-nt siRNAs are produced by DCL4 and DCL2 respectively, and induce PTGS by mRNA cleavage through their association with AGO1
[35, 36]. Interestingly, mRNA cleavage by the 22-nt siRNA containing AGO1 has been reported to result in the recruitment of RDR6, which generates
dsRNA with the 3′ AGO cleavage fragment as a template [4, 40, 41]. The dsRNA is subsequently processed by DCL4 to produce 21-nt siRNAs that
associate with AGO1 and perform PTGS. In the highly repetitive genome of maize, 22-nt siRNAs form a larger proportion of the siRNA total than in A.
thaliana and rice [1, 45]. Surprisingly, it has been suggested that the production of 22-nt siRNAs in maize occurs independently of MOP1, the
homologue of Arabidopsis RDR2 [1, 47]. In contrast, other research suggest that MOP1 does play a role in the production of maize 22-nt siRNAs [60]. In
A. thaliana the majority of siRNAs is 24-nt in length, also referred to as repeat-associated or heterochromatic siRNA, and is mainly produced from
transposons, repeat sequences, and a limited number of pseudogenes [50, 62]. The production of 24-nt siRNAs requires Pol IV [37, 38], which produces
ssRNAs [43]. The ssRNA is converted to dsRNA by RDR2 and cleaved into 24-nt siRNAs by DCL3 [40]. The 24-nt siRNAs usually associate with AGO4, but
can also be incorporated into AGO6 and AGO9 [35]. AGO4 associated 24-nt siRNAs play roles in directing cytosine methylation at DNA sequences
through the process of RNA-directed DNA methylation (RdDM, Box 6).
miRNA and ta-siRNA The biogenesis of miRNAs commences with the production of primary miRNAs (pri-miRNAs) by RNA polymerase II (Pol II)
transcription of miRNA genes, of which the majority is located in intergenic regions [1, 4]. The single strand pri-miRNAs are processed into precursor
miRNAs (pre-miRNA) with hairpin-loop structures by DCL1 [69, 70], as plants do not have Drosha and Pasha orthologues like animals [4]. The pre-
miRNAs are subsequently cleaved by DCL1 into 21-nt double-stranded miRNAs [71]. Following this step the double-stranded miRNA duplexes are
methylated by the RNA methyl-transferase HUA ENHANCER 1 (HEN1), and exported to the cytoplasm [4]. The functional mature miRNA is incorporated
into the AGO protein-centred RISC, most often containing AGO1 [35], but sometimes with AGO7 or AGO10 [1]. The miRNA-AGO-RISC complex is then
able to cause PTGS by slicing mRNA or by inhibiting translation due to the complementarity of mRNA to the miRNA [2, 73, 74].
Ta-siRNAs are 21-nt long small RNAs produced from long dsRNAs [83]. They are derived from the cleavage product of Pol II generated mRNAs
transcribed from TAS loci, after being targeted by certain miRNAs incorporated into AGO1/7-RISC. The mRNA cleavage product is converted into dsRNA
by RDR6 and SUPPRESSOR OF GENE SILENCING 3 (SGS3) [86], and is further processed by DCL4 to produce a phased array of 21-nt siRNAs that are
loaded into AGO1 to interact with target mRNAs and induce PTGS, much like miRNAs [1, 2, 4, 15, 83]. Similar to certain siRNAs, some miRNAs and ta-
siRNAs play roles in TGS through alternative RdDM pathways (Box 6).

- 19 -
Gene expression Several studies that have investigated the genome-wide inheritance patterns of epigenetic
components in F1 hybrids and allopolyploids, as described previously, determined the genome-wide level of gene
expression as well. Most of these studies show that in both F1 hybrids and allo-tetraploids of different species
(rice, maize, and Arabidopsis), gene expression levels are mostly additive, and thus unchanged relative to the
MPV [10, 45, 101, 103, 144, 146]. According to two studies on rice, the number of genes that is transcribed in
hybrid is changed, and shows an increase compared to the parents [88, 147]. In accordance with this, expression
levels of non-additively expressed genes from F1 hybrids and allopolyploids of different species (maize,
Arabidopsis) has been shown by several studies to be mostly above the MPV [45, 103, 144, 146]. Although one
might expect a direct relation between the increased expression of genes in hybrid and allopolyploid plants, and
the increases in vigor related traits displayed by these plants, it is not possible to firmly establish such a relation
based on these findings, as the group of genes with non-additive expression might contain many negative
regulators of transcription. Moreover, is uncertain what proportion of the non-additively expressed genes results
from changes in the epigenetic states of hybrids and allopolyploids. Nevertheless, although the greater majority
of genes in hybrid and allopolyploid plants are expressed in an additive fashion, a substantial number of genes do
display alterations in their transcription level relative to that of the parents and MPV. It is logical to assume that a
part of this group that owes its altered expression to changed epigenetic patterns, probably contributes to the
heterotic phenotype of hybrids and allopolyploids.
Box 5: Histone modifications and chromatin regulation in plants
The addition of histone modifications may have different effects on chromatin structure and expression activity of genes, causing a more open
chromatin state and consequently gene activation, or a more condensed chromatin state and thereby gene repression.
Gene activating histone modifications Histone modifications reported in studies on heterosis, and associated with active gene expression include H3K9
acetylation, H3K4 di- and tri-methylation, H3K9 tri-methylation and H3K36 tri-methylation [3, 4, 13]. These modifications are enriched in the
euchromatic regions of the Arabidopsis genome, and are anti-correlated with nucleosome presence [26]. It has been reported that in 75 % of the genes
in Arabidopsis, mono-, di- or tri-methylation of H3K4 can be found, which is catalysed by the TRX family member ARABIDOPSIS TRITHORAX 1 (ATX1) [4,
28]. The presence of H3K4me1 shows a correlation with methylation of CG motifs, unlike H3K4me2 and H3K4me3 [28, 30]. In contrast to mono- and di-
methylation of H3K9, H3K9me3 is only found at low levels in Arabidopsis [34]. H3K9me3 can be found in euchromatin at TE genes and non-TE genes,
where it has a mildly activating effect on the expression of the latter group [39]; its deposition is catalysed by SU(VAR)3-9 related proteins such as
SU(VAR)3-9 Related 4 (SUVR4) [4, 42]. In Arabidopsis, H3K36 di- and tri-methylation has been shown to be enriched at actively transcribed genes and
catalysed by ASH1 HOMOLOG 2 (ASHH2) [4, 13]. Acetylation of H3K9 is a characteristic modification of non-transposable genes, and there is a positive
correlation between H3K9ac levels and gene expression levels [39]. Strong correlations have been found as well between H3K9ac and H3K4me2/3 at
the same genetic locus near the TSS [26, 28, 51]. H3K56 acetylation is also preferentially found at genes, mainly at the promoters of active genes [13].
Gene repressing histone modifications Histone modifications associated with inactive genes and condensed chromatin include H3K9 di-methylation
and mono-, di- or tri-methylation of H3K27 [3, 26]. The deposition of H3K9me1/2 is catalysed by SU(VAR)3-9 Homologue 2 (SUVH2) and SUVH/4/5/6
[13, 28], and the presence of H3K9me2 has been reported to be highly enriched at repressed transposons, pseudogenes and repetitive sequences [54].
H3K27 methylation is in general a mark of transcriptionally repressed chromatin [4, 13]. H3K27me1 can mainly be found at repressed TEs located in
peri-centromeric regions. At TEs marked with H3K27me1, often H3K9me2 and 5mC are present as well, which further help to repress TE expression;
however, this co-occurrence has not been detected at non-TE genes associated with H3K27me1 [13]. The presence of H3K27me3, whose deposition is
catalysed by the Polycomb Repressive Complexes 2 (PRC2), is almost exclusively restricted to genes that are transcriptionally repressed [4, 13, 39, 61].
Strangely, H3K27me3 in Arabidopsis has been reported to show no correlation with gene expression, in contrast to animals [13, 28].

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Epigenetic states of genes and transposon elements in hybrids and allopolyploids
Genes are responsible for the production of proteins which fulfil a variety of functions ranging from serving as
building material for support tissue, enzymes, transcription factors, etc. Transposons elements on the other hand
affect genome stability, are sources of siRNA production and affect the transcriptional activity of genes. The
epigenetic state of genes and transposon in hybrids and allopolyploids is therefore of prime interest, as
alterations in their epigenetic state may influence the transcriptome, which in the end may contribute to the
typical increase in vigor displayed by hybrid and allopolyploid plants. Changed patterns of epigenetic inheritance
at genes and TEs of hybrids and allopolyploids have indeed been observed in several studies. Greaves et al. (2012)
for example found that in Arabidopsis C24 x Ler hybrids, mC clusters showing non-additive methylation were
enriched (~8.1 %) in gene bodies and flanking regions, while they were underrepresented at TEs [134]. Roughly
13.6 % of these genes displayed a difference in expression relative to the MPV of more than 1.2-fold. Of these
genes with differential expression about 70.4 % (38 of the 54) exhibited an inverse correlation between
methylation levels and gene expression. This does show that changes in gene expression resulting from
alterations in gene methylation do occur in hybrids, although the group of genes for which non-additive
methylation has been found to inversely affect expression, represents only a small fraction of the total of non-
additively methylated genes, suggesting that at most genes non-additive methylation does not affect their
expression. Changed expression of a small group of key genes however, might be sufficient for heterosis to occur.
A different study reported that the same Arabidopsis hybrid had increased DNA methylation relative to their
parents at both protein-coding genes and TEs, and the increase at TEs was greater than in protein-coding genes
[103]. The latter appears to disagree with the findings of Greaves et al. (2012), although it must be noted that the
non-additive methylation reported by Greaves et al. (2012) includes both non-additive decreases and increases,
which have not been specifically described. According to the authors, increased methylation at protein-coding
genes was mainly present in the regions 1kb up- and downstream, as at these regions all C/G sites displayed
elevated methylation levels, while at the coding region the level of CG motifs only was increased. A large
percentage (44 %) of the CG sites differentially methylated between parents and hybrids, and more highly
methylated in hybrids, was located in genic regions, while an even greater percentage (~ 47 %) was located at TEs
[103]. By contrast, only 8 and 11 % of differentially methylated CHG and CHH sites respectively, with higher
methylation levels in hybrids, was located in genic regions, and by far most of these CHG and CHH sites were
located at TEs. TEs are regions of 24-nt siRNA production, which are known to be involved in de-novo methylation
of CHH motifs (Box 3). The observed differential and increased methylation of CHG and CHH sites at TEs might
therefore have resulted from de novo methylation through the RNA-directed DNA Methylation (RdDM) pathway
(Box 3, 6). Also in wheat CG methylation was increased compared to the MPV at TEs of S3 generation
allopolyploids [138], and in rice altered methylation pattern have been identified at genes. In rice hybrids from O.
sativa cv. Nipponbare and cv. 93-11, on average approximately 28.15 % of the genes identified showed
differential DNA methylation between the parents and reciprocal rice NPB x 93-11 hybrids [101]. Roughly 38 % of
the differentially methylated genes was found to display non-additive methylation levels. Moreover, especially
TE-associated genes were often differentially methylated, which indicates that the presence of TEs affects the
epigenetic state of genes and potentially their expression. This suggests that in rice hybrids, like in Arabidopsis a
considerable number of genes may display changed epigenetic states that potentially result in altered gene
expression [101].
Like with DNA methylation, changes in small RNAs, which can potentially result in altered TE and gene regulation,
have been detected in hybrids of different species such as Arabidopsis and wheat [53, 139, 140]. In Arabidopsis
Col x Ler hybrids, roughly 29 % of the differentially expressed small RNA clusters mapped to protein coding genes,
and these small RNA clusters were mostly reduced in expression relative to MPV [139]. However, the largest
group (44 %) of the differentially expressed small RNA clusters mapped to TEs, and was in general expressed at
additive levels [139]. The non-additive reduced expression of mostly genic small RNA clusters suggests that these

- 21 -
genes potentially have changed transcriptional activity with respect to the parents due to altered small RNA
regulation. The authors of this study further stated that a methylated transposon element in the vicinity of a gene
can (negatively) affect the expression of the gene. Knowing that small RNAs can direct DNA methylation, they
investigated whether or not the presence of a TE affected the inheritance pattern of genic small RNA clusters.
They observed that 24 % of genic siRNA clusters without a TE within 1 kb showed additive expression, while 64 %
displayed a LP or BLP pattern [139]. 53 % of genic siRNA clusters with a TE in the vicinity was found to be
additively inherited, and just 32 % demonstrated LP or BLP patterns, once more suggesting that the presence of a
TE affects the epigenetic state of genes. Others have reported that in Arabidopsis Ler x C24 hybrids, most of the
24-nt siRNA clusters mapping to genes and TEs displayed levels similar to the MPV [53]. This finding appear to be
consistent with the mostly additive levels of small RNA cluster at TEs reported above. Most of the tested non-
additive 24-nt siRNA clusters, of which the large majority displayed reduced levels, were associated with genes
and their ﬂanking regions, which might be consistent with the patterns observed for DNA methylation. This,
together with the previous finding on small RNA clusters in Col x Ler hybrids, suggests that in Arabidopsis hybrids,
non-additive reductions in small RNAs and more specifically 24-nt siRNAs, occur mostly at protein coding genes,
while TEs display often additive levels of small RNAs. Additive small RNA levels however, can result in non-
additive DNA methylation resulting from RdDM when small RNA levels are sufficient or insufficient for the
induction of TCM or maintenance of the parental epigenetic state [23]. The de-novo methylation of mainly CHH
sites that might occur in these situations could explain the strong increase in DNA methylation and large
percentage of highly methylated CHH motifs at TEs described above.
Contrasting results have been found in wheat with respect to small RNAs at TEs. In one study, the proportion of
small RNAs matching repeats (mainly TEs) of the total small RNA reads decreased from ~12 % in parents to 6 % in
the first generation allopolyploid of Ae. tauschii and T. Turgidum, with 85 % of them being decreased relative to
MPV [140]. In the wheat hybrid, most (58 %) of the siRNAs corresponding to transposons were non-additively
expressed, with ~67.2 % of them being decreased. This appears to be inconsistent with what has been reported
for Arabidopsis, where small RNAs at TEs display mostly additive levels. According to another study, the
proportion of TE-derived siRNAs relative to the small RNA total increased in S3 allohexaploid compared to both
the diploid parent Ae. tauschii and tetraploid parent T. Turgidum, while the percentage decreased at genic
regions [141], which is consistent with findings in Arabidopsis hybrids. The same study further investigated the
effect of siRNA alterations at TEs on neighbouring genes by looking at one of the parental genomes in the hybrid.
They found that for 97 % of the genes with TEs that display changes in siRNA expression in the close vicinity, the
expression level showed a negative correlation. This suggests that in wheat like in Arabidopsis, the nearby
presence of a TE and its associated epigenetic state has the potential to affect gene expression. In all, there
appears to be considerable evidence for the occurrence of altered epigenetic patterns of genes and TEs, which
has the potential to influence the transcriptome and hence hybrid and allopolyploid phenotypes, either resulting
from alterations in the epigenetic state of the genic regions themselves or the epigenetic state of nearby
repetitive regions and TEs that influence them.

- 22 -
Site specific inheritance patterns of epigenetic modifications and small RNAs, in hybrids and
allopolyploids
In the previous sections we described several observations regarding the genome wide and more region specific
epigenetic states of hybrids and allopolyploid, and possible changes relative to the parental situation. The
observations of at these scales are the cumulative result of local site specific epigenetic states, which is
determined by cis-regulation, and/or trans-effects [98]. Trans-regulation has the possibility to alter the epigenetic
state in the hybrid compared to the parents, whereas cis-regulation will result in a similar epigenetic state as the
parents, dictated by the DNA sequence. As it is of interest to know, with regard to the possible predictability of
these patterns, what apart from the parental DNA sequence determines the site specific pattern of epigenetic
inheritance in hybrids, a number of studies has investigated both the site specific epigenetic state of the parents
as well as that of the hybrid alleles. Sites with similar epigenetic states in both parents have in general been
observed to display no change in the epigenetic state in the hybrid [53, 98]. In intraspecific Arabidopsis hybrids for
example, C/G motifs that were found to be methylated in both parents were in ~95 % also methylated in the
hybrid progeny [98]. Likewise, for C/G sites that were unmethylated in both parents, only ~3.63 % of the sites
displayed methylation in the hybrids. Similar patterns have been reported for siRNAs. Groszmann et al. (2011)
observed that only ~1.36 % of the 24-nt siRNA clusters with no, or a small difference between parents, displayed
non-additive levels in intra-specific Arabidopsis hybrids, of which 80 % showed a reduction relative to the MPV
[53]. This suggests that when the parental epigenetic states are similar, the same pattern is likely to be inherited
in the hybrid, resulting in additive levels. In support of this, additive methylation in Arabidopsis hybrids was found
to be almost exclusively due to parental epialleles retaining their methylation pattern in the hybrid [134].
On the other hand, the same study as described above observed that at C/G sites methylated in only one parent,
only ~50 % was methylated in hybrids [98]. Moreover, according to others, in 84 %, 72 % and 52 % of the regions
that showed differential CG, CHG, and CHH methylation respectively between Arabidopsis parents also showed
differential CG methylation between parents and intraspecific hybrids [103]. This indicates that sites displaying
differential parental methylation are often abnormally inherited in the hybrid Arabidopsis. In contrast to the
findings in Arabidopsis, a study on rice found that most of the genes with differential levels of expression, DNA
methylation and certain histone modifications between the parental alleles, displayed the same allele specific
pattern in the subspecific hybrids, resulting in additive levels [101]. Consistent with this, another study on rice
observed that only ~15 % of the sites that was found to be differentially methylated between parents, was also
found to display differential methylation between hybrids and parents [99]. This percentage is considerably lower
than what has been found in Arabidopsis, and might be attributable to their different genomic composition, as
grasses have an increased genomic GC content relative to eudicots, which does not rely on siRNAs for its
methylation [148]. Nevertheless, a large percentage (~45 %) of the C/G sites differentially methylated between
parents and hybrids was also identified as differentially methylated between the two parents [99]. Yet, based on
these results, the authors suggest that in rice, like in Arabidopsis, sites displaying differential parental methylation
are often abnormally inherited in the F1 progeny, and appear to be the main contributors to non-additive
epigenetic inheritance.
Greaves et al. (2012) studied the allele specific dynamics of cytosine methylation in Arabidopsis hybrids, at alleles
that showed differential methylation between parents and non-additive levels in the hybrid, and observed two
predominant patterns of inheritance. In the ﬁrst, the allele that was highly methylated in one parent retained the
same methylation pattern in the hybrid, whereas the less methylated allele of the other parent displayed an
increase in methylation level, thereby resulting in a non-additive increase in methylation in the hybrid. In the
second pattern, the methylation state of the low methylated parental allele was retained in the hybrid, while the
methylation level of the highly methylated parental allele was reduced in the hybrid, causing a reduction in
methylation in the hybrid [134]. These changes in the methylation level of parental alleles in the hybrid, suggest

- 23 -
that alterations in the hybrid methylation state that often occurs at sites differentially methylated between
parents frequently arise from trans effects that are known as Trans Chromosomal Methylation (TCM) and Trans
Chromosomal de Methylation (TCdM) events, which result in increased respectively decreased methylation levels.
Illustrative for regions with differential parental methylation are the loci At3g43340 and At3g43350 in the
Arabidopsis C24 and Ler ecotypes and their hybrid (Fig. 4a), reported by Greaves et al. (2014). At both loci the Ler
allele acquired the C24 methylation pattern by gaining methylation at CG, CHG, and CHH sites previously
unmethylated in the Ler parent (Fig. 4a), indicating the occurrence of TCM [100]. The acquired mC pattern was
even found to be stably transmitted to all of the F2 genotype combinations (Fig. 4b). At the same loci, H3K9ac
showed an inverse correlation with mC in the parental accessions (Fig. a, b, d), and in the hybrids. H3K9ac levels
followed that of the highly methylated C24 parental allele, which is in agreement with the known correlation
between DNA methylation and histone modifications [54]. Interestingly, TCM at loci At3g43340/50 in the hybrids
had occurred in floral buds but not in the vegetative tissue of 15 DAS seedlings (Fig. b). However, once established
late in the F1 hybrid, the pattern remained present in the F2 throughout the plants life (Fig. b). The unmethylated
Ler parent had higher mRNA levels for each locus compared with the methylated C24 parent (Fig. c), while in F1
seedlings (without TCM) and in F1 floral buds (with TCM) mRNA levels were at MPV and below MPV respectively,
indicating that cytosine methylation and lack of H3K9ac affect expression of the loci. The authors state that TCM
events may not only occur in the hybrid immediately after fertilization, but also at later in plant development.
They further suggest that TCM events like these might be caused by a gradual increase of a “signal” such as siRNA
that triggers RdDM when the concentration surpasses a certain threshold level. Alternatively, TCM might have
occurred in the floral buds due to changes in the epigenetic and chromatin state that take place in cells that are
part of the reproductive cycle (Box 5).
Figure 4: Example of the inheritance of epigenetic components in hybrid and self-cross progeny of A. thaliana C24 and Ler ecotypes. (A) Bisulphite PCR
generated mC patterns in floral buds at loci At3g43340/50 at the parental C24/Ler alleles (up left/right), and C24/Ler alleles of the F1 hybrid (down
left/right). The black asterisk denotes an allele derived from the F1 hybrid. The colours blue, red and green represent cytosine methylation levels at the
motifs CG, CHG and CHH respectively. (B) McrBC qRT-PCR generated mC levels in floral buds (left) and 15 DAS seedlings (right) at loci At3g43340/50 in the

Hybrid vigor in plants, and the role of epigenetics.

Hybrid vigor in plants, and the role of epigenetics.

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