REVIEW

The Genetic Control of Apomixis: Asexual

Seed Formation
Melanie L. Hand and Anna M. G. Koltunow1
Commonwealth Scientific and Industrial Research Organization (CSIRO) Plant Industry,
Glen Osmond, South Australia 5064, Australia

ABSTRACT Apomixis (asexual seed formation) is the result of a plant gaining the ability to bypass the most fundamental aspects of
sexual reproduction: meiosis and fertilization. Without the need for male fertilization, the resulting seed germinates a plant that
develops as a maternal clone. This dramatic shift in reproductive process has been documented in many flowering plant species,
although no major seed crops have been shown to be capable of apomixis. The ability to generate maternal clones and therefore
rapidly fix desirable genotypes in crop species could accelerate agricultural breeding strategies. The potential of apomixis as a next-
generation breeding technology has contributed to increasing interest in the mechanisms controlling apomixis. In this review, we
discuss the progress made toward understanding the genetic and molecular control of apomixis. Research is currently focused on two
fronts. One aims to identify and characterize genes causing apomixis in apomictic species that have been developed as model species.
The other aims to engineer or switch the sexual seed formation pathway in non-apomictic species, to one that mimics apomixis. Here
we describe the major apomictic mechanisms and update knowledge concerning the loci that control them, in addition to presenting
candidate genes that may be used as tools for switching the sexual pathway to an apomictic mode of reproduction in crops.

ITHIN flowering plants (angiosperms), reproduction

W through seeds occurs by sexual and surprisingly, by
asexual pathways. The former generates variation, while
Sexual Seed Formation in Flowering Plants
Seed formation in angiosperms begins with the develop-
mental decision to switch from a vegetative to a reproductive
asexual seed reproduction (apomixis) produces genetically
mode of growth. This manifests with the formation of a flower
identical progeny. (See Table 1 for definitions.) The ability
containing male (anther) and female (ovule) reproductive
to produce genetically identical progeny via seed is of sig-
organs, and within these organs, diploid gamete precursor
nificant value to agriculture for its power to fix complex
cells form (Figure 1). Many diploid male gamete precursor
favorable genotypes, including the higher yields often found
cells differentiate in the anther of the flower; however within
in F1 hybrids. Apomixis is documented in more than 120
the ovule, which is the progenitor of the seed, a single female
angiosperm genera (Carman 1997); however, no major seed
gamete precursor cell differentiates. This cell is called a mega-
crop species are apomictic, and attempts to introduce the
spore mother cell. Both male and female gamete precursor
apomixis trait to crops from apomictic relatives by cross-
cells undergo meiosis followed by mitosis to form the mature
pollination have been largely unsuccessful (Savidan 2000).
multicellular male and female gamete-containing structures,
Apomixis is also of interest from developmental and evolu-
known as the pollen grain and embryo sac, respectively.
tionary perspectives. Most natural apomicts are classified as
Figure 1 illustrates the events leading to the formation of
facultative because they have the ability to reproduce both
the most common form of embryo sac, called the Polygonum-
sexually or asexually. To understand the different pathways
type, which is found in .70% of flowering plants. The diploid
of apomictic seed formation, it is instructive to first examine
megaspore mother cell undergoes meiosis, forming four hap-
the sequence of events required for sexual seed formation.
loid megaspores. One of these megaspores is selected to be-
come the functional megaspore and the others degenerate.
Copyright © 2014 by the Genetics Society of America The haploid functional megaspore then undergoes three mi-
doi: 10.1534/genetics.114.163105 totic divisions without cytokinesis, to produce a coenocyte
Manuscript received February 16, 2014; accepted for publication March 31, 2014
1
Corresponding author: CSIRO Plant Industry, Waite Campus, P.O. Box 350, Glen
containing eight haploid nuclei surrounded by a cell wall
Osmond, SA 5064, Australia. E-mail: [email protected] (stage not shown in Figure 1) (reviewed by Drews and Koltunow

Genetics, Vol. 197, 441–450 June 2014 441

 Table 1 Definitions

Term Definition
Apomixis Plant asexual reproduction through seed. Progeny of an apomictic plant are genetically identical to the
maternal plant.
Embryo sac Multicellular female gamete-producing structure of a flowering plant. Also known as the female
gametophyte.
Apomeiosis Avoidance or failure of meiosis during the development of an embryo sac.
Diplospory Apomeiosis pathway where a diploid embryo sac develops from the megaspore mother cell.
Apospory Apomeiosis pathway where a diploid embryo sac develops from a somatic ovule cell that is not the
megaspore mother cell, called the aposporous initial (AI) cell.
Parthenogenesis Development of an egg found in a diplosporous or aposporous embryo sac into an embryo without
fertilization.

2011). The nuclei migrate and cellularize, producing a seven- embryo initial cells begin mitosis forming multiple globular-
celled mature embryo sac. It contains three antipodal cells shaped embryos that can develop to maturity only if the
whose function is unknown, two synergid cells that have sexually derived embryo sac is fertilized, as the sexual and
roles in attracting the male gametes, a single egg cell, which asexual embryos share the nutrititive endosperm. Sporophytic
is the progenitor of the embryo in the seed, and a large apomixis can therefore lead to a seed containing multiple
central cell containing two nuclei that fuse to form a diploid embryos (Figure 1) and is common in citrus. The sexually de-
precursor nucleus of the endosperm (Figure 1). Seed devel- rived embryo may or may not mature or germinate (Koltunow
opment initiates following double fertilization, where the et al. 1995; Koltunow et al. 1996). Sporophytic apomixis has
male pollen tube containing two sperm cells enters the not been extensively studied at the molecular level; how-
ovule. One sperm cell fuses with the egg cell stimulating ever, it appears genetically complex (García et al. 1999). The
divisions and pattern-forming events that give rise to the remainder of this review focuses on gametophytic apomixis.
diploid embryo. The other sperm cell fuses with the diploid Gametophytic apomixis relates to mechanisms where an
central cell nucleus to initiate divisions to form the nutritive embryo sac is mitotically formed from a diploid cell in the
triploid endosperm, which provides essential resources to ovule, bypassing meiosis. Another term for such mitotic
the developing embryo (Berger et al. 2008). The ovule tis- embryo sac development is apomeiosis. Embryo develop-
sues surrounding the developing embryo and endosperm ment in gametophytic apomixis is fertilization indepen-
contribute to the seed coat in the mature seed. dent whereas endosperm formation may or may not require
fertilization (Figure 1). Apomeiotic embryo sac development
is further subdivided into two types (diplospory and apos-
Apomixis Mechanisms
pory) based upon the origin of the diploid precursor cell that
In contrast to sexual seed formation, apomixis can occur by ultimately gives rise to the mitotically derived embryo sac.
various mechanisms that share three common develop- In diplospory, the precursor is the megaspore mother cell (or
mental components: (i) a bypass of meiosis during embryo a cell with an altered program that differentiates in the
sac formation (apomeiosis), (ii) development of an embryo megaspore mother cell position) (Figure 1). This cell may
independent of fertilization (a process known as parthe- enter meiosis and abort the process or it may immediately
nogenesis), and (iii) formation of viable endosperm either begin mitosis. Diplospory has been observed in species includ-
via fertilization-independent means or following fertiliza- ing Taraxacum officinale (dandelion), Boechera spp., Erigeron
tion with a sperm cell (Koltunow and Grossniklaus 2003). annuus, and Tripsacum dactyloides. By contrast, apospory
Derivation of the egg from a diploid maternal cell without involves development of the embryo sac via mitosis not from
meiotic reduction, and its subsequent fertilization-independent the megaspore mother cell, but from a diploid somatic cell
development into an embryo, means that the progeny derived positioned adjacent to the megaspore mother cell (Figure 1).
from apomictic development are clonal and therefore genet- This cell, termed the aposporous initial cell, undergoes mi-
ically identical to the maternal parent. tosis and the nuclei cellularize. The mitotic events of diplo-
Apomixis mechanisms are historically subdivided into two spory and apospory may or may not make a seven-nucleate
categories and classified as either gametophytic or sporophytic, Polygonum-type embryo sac; however, an egg, a central cell,
based on whether the embryo develops via a gametophyte and synergids are typically formed (Figure 1). Depending on
(embryo sac) or directly from diploid somatic (sporophytic) the species, both sexually derived and aposporous embryo
cells within the ovule (Figure 1; Nogler 1984; Koltunow sacs can coexist within the one ovule, as occurs in Brachiaria
1993). During sporophytic apomixis, development of an em- species (Araujo et al. 2000). Alternatively, development of the
bryo sac following the typical angiosperm sexual pathway aposporous embryo sac may lead to the demise of the sexu-
still occurs. However, during mitosis of the functional mega- ally derived embryo sac or pathway as occurs in aposporous
spore, diploid somatic ovule cells surrounding the embryo Hieracium and Pennisetum species (Peel et al. 1997; Koltunow
sac differentiate and have an embryogenic cell fate. These et al. 2011). Embryo development from the diploid egg formed

442 M. L. Hand and A. M. G. Koltunow

Figure 1 Mechanisms of sexual and apomictic seed development. Seed developmental processes occur within the ovule of the flower, which is depicted
as a single floret typical of Hieracium species in this figure. This diagram compares the major differences in the seed development pathway for sexual
seed formation and the apomictic mechanisms of sporophytic and gametophytic apomixis. Meiosis, mitosis, and double fertilization constitute the major
components of the seed formation pathway. Arrows passing through each of these components represents the involvement of a given component
within a particular pathway. In the process of gametophytic apomixis, embryo sac formation can occur via either apospory or diplospory, which are
distinguished by different embyro sac precursor cells. In gametophytic apomixis, embryo formation is initiated in the absence of fertilization (parthe-
nogenesis); however, endosperm formation can occur either with or without fertilization, which is represented by a dashed line. The relative ploidy level
of cells (n) is tracked for various components throughout each pathway. The ploidy level of endosperm formed through gametophytic apomixis is
variable, depends on a number of factors, and is therefore represented by a question mark (?). In the depicted apospory pathway, the sexual pathway is
shown to terminate once the aposporous initial cell undergoes mitosis. Different colors track the precursor cells that form the embryo for each pathway:
sexual (white), sporophytic apomixis (green), diplospory (yellow), and apospory (blue).

Review 443
in aposporous and diplosporous embryo sacs occurs without meiosis and as aposporous initial cells are not formed,
fertilization and the term parthenogenesis is used to describe a functional haploid embryo sac develops, and the egg and
this process (Figure 1). Endosperm development can occur central cell form an embryo and endosperm, respectively, in
without fertilization of the central cell, although this is rare, the absence of fertilization. Deletion of both the LOA and
occurring predominantly in members of the daisy family LOP loci results in complete reversion to sexual development
(Asteraceae). Apomicts that require fertilization to produce (Catanach et al. 2006; Koltunow et al. 2011). Apomixis in
endosperm have disturbed maternal and paternal genome aposporous Hieracium therefore seems to be superimposed
contributions (m:p) in the endosperm. For example, in such on the sexual pathway, suggesting that apomixis may redi-
apomicts, fertilization of a tetraploid central cell may lead to rect the fate of cells with gametic potential, rather than
a 4m:1p endosperm genome ratio, in contrast to the typical being a detached and completely independent pathway
2m:1p ratio of fully sexually reproducing species. Those apo- (Koltunow et al. 2013). This theory is consistent with the
micts that require fertilization to develop endosperm have known coexistence of apomixis and sex, suggesting that if
therefore developed multiple strategies to ensure seed viability the apomixis pathway is not initiated in an ovule, the sexual
and these have been discussed in previous reviews (Koltunow pathway remains functional. One interpretation is that apo-
and Grossniklaus 2003; Curtis and Grossniklaus 2008). mixis may be thought of as an alteration to the spatial and
temporal expression of sexual reproductive processes, where
the sexual pathway is initiated at a different time and/or in
Genetics and Inheritance of Apomixis
a different cell. For diplospory, the megaspore mother cell
The presence of varied apomictic mechanisms and the phylo- appears to be specified with gametic potential; however, the
genetic positioning of apomictic species throughout many sexual pathway is altered so that the events of meiosis do
angiosperm families together imply that apomixis has evolved not initiate, or meiosis is altered so that it is not successfully
independently multiple times (Carman 1997; Van Dijk and completed. For apospory, an altered sexual pathway appears
Vijverberg 2005). Genetic analyses using apomicts as pollen to be expressed in additional cells within the ovule, which
donors in crosses with sexual individuals as maternal parents results in diploid somatic cells mitotically forming an em-
have shown apomixis to be inherited as a dominant trait. bryo sac, which is usually the role of the haploid functional
Early genetic studies proposed that a single dominant locus megaspore in sexual reproduction. During the develop-
controlled apomixis in most studied apomictic species. For ment of the diploid diplosporous and aposporous embryo
some species, it has since been revealed that many apomixis sacs, the egg cell and central cell differentiate with a capa-
loci exhibit suppressed recombination. Studies involving genetic bility to directly transition into an embryo and endosperm,
analyses searching for rare recombinants in suppressed regions respectively, if seed formation is completely fertilization
and gamma deletion mutagenesis have revealed that in some independent. Studies using various reproductive markers
species, the developmental components of apomixis (meiotic support these hypotheses (Tucker et al. 2003; Rodrigues
avoidance, parthenogenesis, and fertilization-independent et al. 2010).
endosperm development) are controlled by independent loci. Most apomicts are polyploid, and apomixis has previously
For instance, in Taraxacum and Erigeron species, two inde- been proposed to be a consequence of hybridization and/or
pendent loci have been identified that control diplospory and genome doubling, i.e., the events of polyploidization (Carman
parthenogenesis (Van Dijk et al. 1999; Noyes and Rieseberg 1997). However, polyploidization alone is not sufficient to
2000). Similarly, apospory and parthenogenesis are controlled induce apomixis, as not all polyploids are apomicts. Apo-
by two independent loci in Hypericum, Poa, Hieracium, and mixis has previously been considered as an evolutionary dead
Cenchrus species (Albertini et al. 2001; Catanach et al. 2006; end, as it is associated with loss of the ability to generate
Schallau et al. 2010; Conner et al. 2013). Genetic studies in genetic variation through recombination, which renders apo-
Hieracium, which is also capable of fertilization-independent mictic populations incapable of adapting to environmental
endosperm formation, have revealed that this trait can also change. However, the incomplete penetrance of apomixis
segregate independently of the other two apomictic compo- and maintenance of a normal sexual pathway in most apo-
nents (Ogawa et al. 2013). micts provides opportunity for genetic diversification and
Interestingly, characterized gamma deletion mutants have evolution via sexual recombination (Chapman et al. 2003;
revealed that sexual reproduction is the default pathway in Hörandl and Hojsgaard 2012).
apomictic Hieracium praealtum. A series of deletion mutants
that lack the apospory locus (called LOSS OF APOMEIOSIS
Characteristics of Apomixis-Related Loci
or LOA) and an additional locus responsible for fertilization-
independent seed development (called LOSS OF PARTHENO- Attempts to identify apomixis loci through map-based cloning
GENESIS or LOP) were developed. Deletion of either LOA or approaches have proven challenging in many apomictic species,
LOP alone sees the return of the sexual pathway for that as recombination is often suppressed around apomixis loci. This
component. For example, plants with LOA deleted no longer distortion of recombination frequency at apomixis loci suggests
produce diploid embryo sacs via apospory as they lack apo- strong divergence of alleles at the apomixis-related geno-
meiosis function. Instead, the megaspore mother cell undergoes mic regions. In the most extreme cases, the divergence has

444 M. L. Hand and A. M. G. Koltunow

been associated with hemizygosity of the associated loci e.g., ing of these loci has revealed a number of genes with the
Hieracium (Okada et al. 2011), Pennisetum (Akiyama et al. potential to have critical roles in apomixis.
2005), and Paspalum (Calderini et al. 2006). Some apomixis The ASGR of Pennisetum was originally identified following
loci have also been associated with heterochromatin and/or random amplified polymorphic DNA (RAPD)-based analysis of
substantial repetitive sequences. In apomictic Pennisetum a selection of apomictic and sexual plants, which revealed a
squamulatum, the apospory-specific genomic region (ASGR) set of apomixis-specific markers that define the ASGR (Ozias-
is located at a heterochromatic and nonsynaptic telomeric Akins et al. 1998). Shotgun sequencing of Pennisetum and
region of a single chromosome (Akiyama et al. 2004). Chro- Cenchrus bacterial artificial chromosome (BAC) clones from
mosomes carrying the ASGR in Pennisetum and the LOA locus the ASGR identified 40 putative protein coding regions, two
in Hieracium are associated with extensive repetitive sequence of which had sequence similarity to the rice BABY BOOM
and transposon-rich regions (Akiyama et al. 2004; Okada et al. (BBM) gene (Conner et al. 2008). BBM was originally identified
2011). Similarly, the hemizygous apomictic controlling locus in Brassica napus as an AP2-domain transcription factor, and
(ACL) of Paspalum, which shows strong suppression of recom- overexpression in Arabidopsis results in embryo development
bination, has undergone large-scale rearrangements due to from vegetative tissue (Boutilier et al. 2002). The ASGR–BBM-
transposable elements, when compared to a syntenic region like genes, which are present in the ASGR of both Pennisetum
in rice (Calderini et al. 2006). These commonalities of re- and Cenchrus, are therefore candidate genes with strong po-
petitive, heterochromatic regions associated with apomixis tential to have a role in the induction and/or maintenance of
loci have led to suggestions that such a repetitive chromo- apomixis events (Conner et al. 2008).
somal structure may have a functional role in apomixis. For In Hypericum, the HYPERICUM APOSPORY (HAPPY) locus
example, one hypothesis is that the repetitive sequences may was identified following an amplified fragment length poly-
act as a sink to sequester factors involved in the sexual repro- morphism (AFLP)-based screen of a selection of apomictic
ductive pathway, thereby altering the expression of sexual re- and sexual plants. When an AFLP, which perfectly cosegre-
productive processes, and possibly causing apomixis (Koltunow gated with apospory, was used to screen a Hypercium BAC
and Grossniklaus 2003). However, recent results suggest that library, a single clone that contained, among other genes,
the abundant repetitive sequences on a chromosome carrying a ubiquitin-mediated E3 ligase was identified (Schallau et al.
the Hieracium apospory locus (LOA) are not required for apos- 2010). This ARIADNE 7-like E3 ligase (HpARI) has been pos-
pory to successfully occur. In this study, progeny arising from tulated as a strong candidate for the HAPPY locus, as one of
a cross between sexual H. pilosella and apomictic H. praealtum the four HpARI alleles within the tetraploid apomict is trun-
that are recombinant for LOA-linked markers are missing cated compared to sexual plants. The truncated HpARI gene
the extensive repetitive sequence structure associated with may act in a dominant-negative fashion, interacting with
the locus, yet still remain apomictic (Kotani et al. 2013). If the the three remaining intact alleles. E3 ligases are involved in
repetitive chromosomal structure associated with some apo- ubiquitin-mediated protein degradation and are known to
mixis loci is not required for function of the locus, it is possi- have a role in embryo sac development. For instance, the
ble that these structural features and allele divergence are MATH-BTB protein MAB interacts with an E3 ubiquitin ligase
a consequence of asexual reproduction and suppressed re- component (Cullin 3a) and is required for mitotic spindle func-
combination, which may have evolved to maintain genic ele- tion and nuclear fate in developing maize (Z. mays) embryo
ments required for apomixis. The chromosomal structure and sacs (Juranić et al. 2012). Alteration in expression or function
degree of recombination may reflect the evolutionary age of of the HpARI E3 ligase might therefore affect embryo sac de-
apomixis in a given species. For instance, those species for velopment in Hypericum, leading to apomixis events.
which the apomixis components can be separated by recom-
bination are likely to be evolutionarily recent apomicts [e.g.,
Differentially Expressed Genes Associated
Poa (Albertini et al. 2001), Taraxacum (Vijverberg et al.
with Apomixis
2010), Hypericum (Schallau et al. 2010), Erigeron (Noyes
and Rieseberg 2000), and Panicum (Kaushal et al. 2008)]. Next-generation sequencing has fueled gene expression-
By contrast, evolutionarily older apomicts are likely to be those based approaches in a range of apomictic species, the reasoning
characterized by apomixis associated loci found in hetero- being that gene transcripts increased or decreased in abun-
chromatic regions that display suppressed recombination dance in apomicts relative to sexual individuals will provide
[e.g., Pennisetum (Ozias-Akins et al. 1998), Brachiaria (Pessino clues to the differential transcriptional pathways involved in
et al. 1998), Paspalum (Labombarda et al. 2002), and Tripsacum apomixis. If apomixis is a consequence of spatial and temporal
(Grimanelli et al. 1998)]. changes in expression of sexual pathway-related genes,
comparative gene expression provides a method with which
to identify such genes. This approach is complicated by the
Genes Linked with Apomixis Loci
nature of asexual reproduction that prevents the generation
Despite the suppressed recombination observed in some of isogenic lines that differ solely with regard to reproductive
apomictic species, loci genetically linked to components of mode. Nevertheless, transcriptome comparisons between apo-
apomixis have been identified in various species, and sequenc- mictic and closely related sexual individuals from reproductive

Review 445
tissues at different developmental stages have uncovered death and the degeneration of the nonfunctional megaspores,
pathways potentially central to apomixis including protein with one of the two isolated copies (APOSTART1) overexpressed
degradation, transcription, stress response, and cell-to-cell in sexual lines relative to apomicts. The Arabidopsis APOSTART1
signaling (Albertini et al. 2005; Laspina et al. 2008; Sharbel ortholog is expressed in mature female embryo sacs and devel-
et al. 2010; Silveira et al. 2012; Okada et al. 2013). oping embryos, and the phenotype of APOSTART1/APOSTART2
It is hoped that comparative gene expression studies in double mutants suggests that this gene has a role in embryo and
aposporous plants may reveal genes and pathways involved seed development (Barcaccia and Albertini 2013).
in processes such as cell-fate specification of the aposporous Within diplosporous species, comparative gene expres-
initial cell, reprogramming of a somatic cell to a germ cell sion studies performed during embryo sac development may
fate, and degradation of sexual products (e.g., the megaspores). identify genes that allow the megaspore mother cell to avoid
In the aposporous grass Brachiaria brizantha, low-depth or modify meiosis. Few transcriptome studies comparing
expressed sequence tag (EST) libraries made from ovaries diplosporous and related sexual taxa have been reported,
of sexual and apomictic individuals revealed ESTs that show although Selva et al. (2012) identified genes differentially
similarity to genes with known involvement in female expressed between diplosporous and sexual Eragrostis curvula,
embryo sac development (Silveira et al. 2012). Further which include retrotransposon proteins, but no obvious can-
characterization of some of these ESTs identified genes didates known to affect meiosis. Sharbel et al. (2010) com-
that are expressed in the region where aposporous initial pared gene expression between apomictic and sexual Boechera
cells appear. These genes include a helicase (BbrizHelic), species across four stages of development to investigate whether
a MADS-box transcription factor (BbrizAGL6), and a stress- a heterochronic change in gene expression is the mechanism
induced protein (BbrizSti1), suggesting that stress-related by which a diploid embryo sac can develop from the mega-
genes may play a role in the induction of aposporous initial spore mother cell in a sexual background. This study reported
cells (Silveira et al. 2012; Guimarães et al. 2013). In apospo- a global decrease in gene expression within the apomict com-
rous Hieracium praealtum, stress-like genes are also expressed pared to the sexual species during the early stages of devel-
in individual aposporous initial cells and early stage embryo opment. While not identifying any individual candidate
sacs that contain two to four nuclei (Okada et al. 2013). In situ genes, the authors hypothesize that apomeiosis is correlated
hybridization also reveals that at least three stress-associated with a global downregulation of gene expression during mega-
genes are upregulated in the aposporous initial cell, but are spore mother cell formation.
undetectable in sexual ovules. It is plausible that stress-
associated pathways may be involved in cell-to-cell signaling
during the appearance of aposporous initial cells, or possibly Knowledge Gained from Sexual Species
in the degeneration of cells derived from the sexual pathway. If apomixis represents an alteration in the spatial and temporal
Components related to the ubiquitin-mediated proteasome expression of the sexual pathway and recruits genes involved
pathway were also identified as enriched in aposporous initial in sex, it follows that conversion of the sexual pathway to an
cells and the early stage aposporous embryo sac when com- apomictic one in nonapomictic species should be possible. This
pared to other somatic ovule cells (Okada et al. 2013). This assumption is supported by the characterization of sexual
finding, combined with the identification of the HpARI E3 mutants that mimic different aspects of apomixis, thus pro-
ubiquitin ligase within the Hypericum apospory locus, makes viding candidate genes that may be involved in apomixis.
the ubiquitin-mediated pathway a candidate for involvement Recent reviews have described many of these genes and
in aposporous initial cell initiation and growth. provide comprehensive lists of genes and mutants that show
Other gene expression studies in aposporous species have apomictic phenotypes (Barcaccia and Albertini 2013; Koltunow
used cDNA–AFLPs to identify differentially expressed tran- et al. 2013).
scripts. In Poa pratensis, this led to the identification and
Mutants that develop embryo sacs without meiosis
characterization of two candidate genes: SOMATIC EMBRYO-
(apomeiosis phenotype)
GENESIS RECEPTOR-LIKE KINASE (PpSERK) and APOSTART
(Albertini et al. 2004; Albertini et al. 2005). Both PpSERK and Mutants that display a phenotype reminiscent of diplospory
APOSTART were isolated in two copies, although further cop- involve genes that affect meiosis. For example, the maize
ies or alleles are present within the P. pratensis genome. elongate1 mutant produces functional diploid embryo sacs
PpSERK is a tyrosine kinase proposed as the switch that by skipping meiosis II (Barrell and Grossniklaus 2005). Sim-
allows the aposporous initial cell to form and develop into ilarly, the dyad mutant of Arabidopsis can produce diploid
an embryo sac. In apomictic P. pratensis, PpSERK is expressed embryo sacs following disrupted meiosis, where only a single
in cells neighboring the megaspore mother cell, which is the equational division occurs followed by an arrest in progres-
region where aposporous initial cells appear (Albertini et al. sion (Ravi et al. 2008). Three recessive Arabidopsis mutants
2005). This suggests PpSERK may play a role in specifying an that have altered meiotic processes have been identified
embryo sac fate to somatic cells. APOSTART contains a lipid- and, when combined in a triple mutant, are able to replace
binding START domain and is believed to have a role in meiosis with mitosis, mimicking the apomeiosis phenotype.
meiosis. APOSTART may also be related to programmed cell This triple mutant, called MiMe (Mitosis instead of Meiosis)

446 M. L. Hand and A. M. G. Koltunow

combines mutants in the omission of second division 1 (Osd1), between apomictic and sexual reproduction may be epigenet-
Atspo11-1, and Atrec8 genes and produces viable diploid game- ically regulated via alterations in chromatin state.
tes that are genetically identical to their mother (D’Erfurth In maize, mutation of AGO104 similarly results in an
et al. 2009). The MiMe and dyad mutants have both been used apomixis-like phenotype. However, the ago104 phenotype
in an attempt to engineer apomixis in Arabidopsis as a proof of mimics diplospory, with a single megaspore mother cell
concept (Marimuthu et al. 2011). Instead of replicating par- per ovule undergoing mitosis rather than meiosis, resulting
thenogenesis, MiMe and dyad mutants were crossed with an in diploid gametes (Singh et al. 2011). AGO104 protein
Arabidopsis line that contains a manipulated centromere- accumulates in somatic cells surrounding the megaspore
specific histone (CENH3), which has previously been dem- mother cell, implying that a mobile signal may be responsi-
onstrated to cause genome elimination (Ravi and Chan 2010). ble for repressing a somatic fate in germ cells. AGO genes
These combinations resulted in diploid progeny that were have also been implicated in germ-cell identity in rice. For
genetically identical to the maternal parent, as the paternal example, MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1) is an
genome had been eliminated. While the outcome of this ARGONAUTE gene expressed specifically in gamete precur-
synthesis is the same as in apomixis, crossing is still required. sor cells within ovules of rice and is essential for normal
Nevertheless, this elegant system clearly demonstrates the sporogenesis and meiosis in these cells (Nonomura et al.
feasibility of engineering clonal propagation by seeds into 2007).
sexual species. Following the characterization of these mutants and the
Specification of the functional megaspore is known to be subsequent hypothesis that apomixis may be epigenetically
affected by both cytokinin and auxin signaling in Arabidopsis, regulated, approaches that compare expression of sRNAs in
suggesting that both these signaling pathways may be impor- natural apomicts to relative sexual plants have begun, although
tant in aposporous initial cell initiation. Arabidopsis cytokinin have not yet yielded causal links between sRNAs and apomixis
receptor triple mutants show disrupted embryo sac develop- (Amiteye et al. 2011; Galla et al. 2013). Given the accumulation
ment. The absence of a functional megaspore-specific marker of evidence supporting the potential epigenetic regulation of
in mutant ovules suggests that disruption of cytokinin signal- apomixis obtained from sexual mutants, studies exploring dif-
ing blocks functional megaspore specification (Cheng et al. ferential large RNA expression combined with changes in small
2013). Similarly, the auxin efflux carrier gene PIN-FORMED 1 RNA are likely to provide important insights into the genetic
(PIN1) is necessary for embryo sac development in Arabidopsis regulation of apomixis.
(Ceccato et al. 2013). Moreover, inhibition of polar auxin It is possible that apomixis represents reversible, epige-
transport in Hieracium also affects the timing, position, and netic silencing of the sexual pathway. Epigenetic regulation of
frequency of aposporous initial cell formation (Tucker et al. apomixis is an attractive hypothesis as it potentially accounts
2012). Alterations in cytokinin and/or auxin signaling may for the facultative nature of apomixis and the ability of
therefore be important in specifying an embryo sac identity to apomicts to revert to sexuality. In an evolutionary sense, it
aposporous initial cells of aposporous plants. may be that such epigenetic changes occur following poly-
Genes with a known role in epigenetic regulation have ploidization and subsequently activate the expression of
been implicated in the regulation of ovule cell-fate specifica- apomixis and suppression of sexual reproduction. In this
tion and embryo sac development. Mutation of ARGONAUTE scenario, all angiosperms may be considered as having
genes within sexual plants can lead to changes in the number “potential” for apomixis (Hörandl and Hojsgaard 2012).
of cells that have the capacity to initiate embryo sac develop-
Mutants capable of fertilization-independent embryo
ment (Olmedo-Monfil et al. 2010; Singh et al. 2011). ARGO-
and endosperm development
NAUTE (AGO) proteins carry small RNAs (sRNAs) forming
complexes with other proteins that cleave mRNA or lead to Genes that may be involved in either fertilization-independent
RNA dependent DNA methylation (Meister 2013). In Arabi- embryo or endosperm development have also been identified
dopsis, ago9 mutants appear to form multiple cells with the through sexual mutants. Interestingly, many of these genes are
potential to mitotically form embryo sacs within a single associated with the Polycomb-group (PcG) chromatin model-
ovule, a phenotype reminiscent of apospory (Olmedo-Monfil ing complex. In particular, the Polycomb repressive complex 2
et al. 2010). Similar phenotypes are also observed in other (PRC2) is known to have a role in suppressing seed devel-
sRNA pathway mutants including rna-dependent rna polymer- opment in the absence of fertilization. The PRC2 is conserved
ase 6 (rdr6) and suppressor of gene silencing 3 (sgs3) (Olmedo- between plants and animals and is responsible for repressing
Monfil et al. 2010). Inactivation of DNA methyltransferases gene expression via trimethylation of histone H3 at lysine
DMT102 and DMT103 in maize also results in apomictic-like 27 (H3K27me3). When core PRC2 genes are mutated in
phenotypes, including the production of diploid gametes and Arabidopsis, phenotypes of fertilization-independent seed de-
multiple embryo sacs within a single ovule, instead of the velopment have been observed. For example, the fertilization-
usual single embryo sac (Garcia-Aguilar et al. 2010). These independent seed (FIS) PRC2 complex (FIS-PRC2) consists of
results suggest that DNA methyltransferases may have a role in the genes MEDEA (MEA), FIS2, FERTILIZATION-INDEPENDENT
the cell-fate specification of embryo sac precursor cells. These ENDOSPERM (FIE), and MULTICOPY SUPPRESSOR OF IRA1
apomixis-like phenotypes further suggest that differentiation (MSI1). Loss of function of any of these genes results in

Review 447
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