Ishii 2016

PP67CH10-Houben ARI 7 January 2016 14:17
Review in Advance first posted online

V I E W
E on January 15, 2016. (Changes may
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still occur before final publication
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online and in print.)
C E
I N
A
D V A
Haploidization via
Chromosome Elimination:
Means and Mechanisms
Access provided by La Trobe University - Bendigo on 01/17/16. For personal use only.
Takayoshi Ishii, Raheleh Karimi-Ashtiyani,

Annu. Rev. Plant Biol. 2016.67. Downloaded from www.annualreviews.org
and Andreas Houben

Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), 06466 Stadt Seeland,
Germany; email: houben@ipk-gatersleben.de
Annu. Rev. Plant Biol. 2016. 67:10.1–10.18 Keywords

The Annual Review of Plant Biology is online at centromere, CENH3/CENPA, haploid, plant breeding
plant.annualreviews.org
This article’s doi: Abstract

10.1146/annurev-arplant-043014-114714
The ability to generate haploids and subsequently induce chromosome dou-
Copyright c 2016 by Annual Reviews. bling significantly accelerates the crop breeding process. Haploids have
All rights reserved
been induced through the generation of plants from haploid tissues (in situ
gynogenesis and androgenesis) and through the selective loss of a parental
chromosome set via inter- or intraspecific hybridization. Here, we focus
on the mechanisms responsible for this selective chromosome elimination.
CENH3, a variant of the centromere-specific histone H3, has been exploited
to create an efficient method of haploid induction, and we discuss this ap-
proach in some detail. Parallels have been drawn with chromosome-specific
elimination, which occurs as a normal part of differentiation and sex deter-
mination in many plant and animal systems.
10.1
Changes may still occur before final publication online and in print
Contents
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2
THE GENERATION OF HAPLOIDS VIA WIDE HYBRIDIZATION . . . . . . . . . . . 10.2
THE ELIMINATION OF A MICRONUCLEATED GENOME . . . . . . . . . . . . . . . . . . 10.5
CENH3 BEHAVIOR IN STABLE HYBRIDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5
GENERATION OF HAPLOIDS USING CROSSES WITH INTRASPECIFIC
HAPLOID INDUCER LINES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6
GENERATION OF HAPLOIDS USING TARGETED
CENTROMERE MANIPULATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.7
CHROMOSOME-TYPE-SPECIFIC ELIMINATION AS PART OF CELL
DIFFERENTIATION AND SEX DETERMINATION . . . . . . . . . . . . . . . . . . . . . . . .10.11
SEX DETERMINATION VIA CHROMOSOME ELIMINATION . . . . . . . . . . . . . . .10.11

ELIMINATION OF GERMLINE-RESTRICTED CHROMOSOMES . . . . . . . . . . . .10.12
CONCLUDING REMARKS AND OUTLOOK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.12
INTRODUCTION
The ability to generate haploids and subsequently induce chromosome doubling has provided a
strategy to significantly accelerate the crop breeding process. The major advantage to breeders
of such doubled haploids (DHs) lies in the simultaneous fixation at every locus within a single
generation, which avoids the time-consuming conventional requirement for extensive selfing or
backcrossing before true-breeding lines can be obtained. Haploids have been induced through the
generation of plants from haploid tissues (in situ gynogenesis and androgenesis) and through the
selective loss of a parental chromosome set via inter- or intraspecific hybridization. The choice of
method for generating haploids is rather species dependent (for reviews, see 15, 16, 21). Once a
DH line has been created, its genotype can be rapidly multiplied, allowing breeders to evaluate
the many quantitative traits they need to handle when developing a superior cultivar. The result
is substantial savings in both time and resources over conventional practice. The value of haploids
has also been recognized in the context of genetic studies at the cellular level, and their use has
Haploid: an organism
that harbors only a started to extend beyond plants to animal systems (for a review, see 85). The present review focuses
single copy of each on the mechanisms underlying the selective elimination of one of the genomes during the early
homolog in its somatic cell divisions of a hybrid embryo and describes in some detail methods for haploid induction that
cells; the term can also involve targeted centromere manipulation.
refer to the cells
themselves
THE GENERATION OF HAPLOIDS VIA WIDE HYBRIDIZATION
Doubled haploid
(DH): the product of The outcome of gamete fusion in a wide hybrid is the union of two distinct genomes within a
a chromosome- single nucleus, with the cytoplasmic DNA generally being exclusively of maternal origin. Such
doubled haploid; the novel genomic combinations are typically unstable and are subject to a high degree of both genetic
doubling can be either and epigenetic reorganization (77). The complete elimination of one of the parental genomes is
spontaneous or
not uncommon: It has been documented in 74 hybrids involving monocotyledonous species and 35
induced by various
treatments involving dicotyledonous species (Supplemental Table 1; follow the Supplemental Materials
link from the Annual Reviews home page at http://www.annualreviews.org).
Haploidization: the
transformation of a Producing wide hybrids by intercrossing monocotyledonous species requires an embryo rescue
diploid into a haploid intervention because the hybrid endosperm often aborts. The process of haploidization follow-
ing uniparental genome elimination was first investigated in hybrids between cultivated barley
(Hordeum vulgare) and its wild relative Hordeum bulbosum, a perennial allogamous species belonging
10.2 Ishii · Karimi-Ashtiyani · Houben
to barley’s secondary gene pool. Davies (11) was able to obtain three barley-like diploid progeny
from a cross between 4x H. bulbosum as the female and 4x H. vulgare as the male and suggested
that these progeny originated from male parthenogenesis. Only later did studies confirm that the
CENH3: the
underlying mechanism is actually chromosome elimination (51, 52, 88). The similar absence of centromere-specific
H. bulbosum chromosomes in the karyotype of progeny of crosses between 2x H. vulgare and 2x histone H3 variant
H. bulbosum (44) led to the development of the so-called bulbosum method, which in conjunction homologous to
with embryo rescue was able to generate haploid barley embryos in up to 30% of pollinated florets CENP-A, CID,
Cse-4p, and HTR12;
(for a review, see 49). This approach was later used to generate haploids in other species (see
it is present in the
Supplemental Table 1). kinetochore complex
The success of haploid barley formation through uniparental chromosome elimination de- of active centromeres
pends on several genetically unlinked factors (33). Higher ambient temperatures during the early
stages of embryo growth tend to promote chromosome elimination (67). H. bulbosum chromo-
somes are eliminated from the hybrid embryo several days after fertilization (4) independent of
the crossing direction, although for some genotypes, hybrids combining both parental sets of
chromosomes can also be recovered (36). In the combination Hordeum marinum × H. vulgare,
the barley genome was eliminated in the endosperm, but the H. marinum one was eliminated in
the embryo (19). Two proposed sources of uniparental chromosome elimination are a difference
in the timing of mitosis (29) and asynchrony in nucleoprotein synthesis (4, 54); others include
the formation of multipolar spindles, different genome ratios (86), a spatial separation between
genomes at interphase (20) and/or at metaphase (83), the selective inactivation of centromeres
(19, 63, 79), and—analogous to the host-restriction and host-modification systems exhibited by
certain bacteria (5)—the degradation of one set of chromosomes through nuclease activity (12).
Lange & Jochemsen (53) have suggested that the suppression of nucleolar activity in the genome
undergoing elimination increases its susceptibility to whatever factor is inducing elimination.
The pioneering cytological study of the elimination process revealed that chromosome loss is
associated with the formation of micronuclei during mitosis during the first few cell divisions in
the hybrid embryo. Chromosomes destined for elimination appeared to not congregate properly
at metaphase and lagged at anaphase (54). Micronuclei have long been known to reflect the
enclosure of lagging chromosome fragments during the reconstitution of the nuclear membrane
after mitotic telophase. More recently, the recognition within the H. vulgare × H. bulbosum
embryo of a centromeric histone H3 variant known as either CENH3 or CENP-A has implied
that uniparental centromere inactivation determines chromosome elimination (79) (Figure 1). In
unstable hybrids, active centromeres label positively for CENH3, whereas inactive (H. bulbosum)
centromeres attract little or no label. Sperm-derived centromere-incorporated CENH3 likely
provides residual kinetochore function for the H. bulbosum chromosomes, but when its amount
falls below a critical threshold, chromosome segregation fails and the chromosome is eliminated.
That a limited fraction of parental H3 variants is transmitted to the progeny has been demon-
strated in animal embryos (35). If preexisting CENH3 is partitioned equally between duplicated
sister centromeres and no de novo incorporation of CENH3 into H. bulbosum centromeres occurs,
its titer should be approximately halved at each cell division. In humans, as little as 10% of the
endogenous CENH3 level is sufficient to support kinetochore assembly (56). If a zygotic resetting
of CENH3, as demonstrated in Arabidopsis thaliana (38), also occurs in Hordeum, then the active
removal of both parental CENH3s prior to the first zygotic division should occur, thereby
diminishing the possibility of reactivating the H. bulbosum centromeres in an unstable hybrid.
In unstable H. vulgare × H. bulbosum hybrids, the uniparental transcription inactivation of
CENH3 is not the cause of centromere inactivation (79). The presumption is that, instead, CENH3
is not successfully incorporated into the centromeres of the H. bulbosum chromosomes. The regu-
lation of CENH3 loading and assembly into the centromere is mediated by multiple proteins, the
www.annualreviews.org • Haploidization via Chromosome Elimination 10.3
Unstable interspecific cross Stable interspecific cross

1 CENH3 CENH3 1 CENH3 CENH3
Somatic
parents
Somatic
Diploid
parents
Diploid
cells
cells
2 Sperm cell
2 Sperm cell
Egg cell Egg cell
Gametes
Gametes
× ×
3 3
zygote
zygote
Early
Early
4 Mitosis Mitosis 4
dependent independent
i ii iii
Embryogenesis
Embryogenesis
iv
5 5
Haploid Hybrid
Female CENH3-positive centromeres Micronucleus derived from lagging chromosome

because of centromere inactivity, sister chromatid
Male CENH3-positive centromeres mis-segregation, or budding of chromatin
Centromeres with residual amounts of CENH3 x Micronucleus undergoing degeneration
Nuclear genomes Maternal cytoplasm
Paternal cytoplasm
Figure 1
A mechanistic model for uniparental chromosome elimination in unstable wide-hybrid embryos (left) compared with embryo formation
in stable hybrids (right). () The process starts with a pair of diploid parents, each of which encodes its own CENH3. (, ) Next, a
hybrid zygote forms by a wide cross. (, ) In stable hybrids, no chromosomes are eliminated. The behavior of parental CENH3s
during early embryogenesis is unknown; the centromere sequences of the parental chromosomes may be able to process either both
forms or only one form of CENH3. In unstable hybrids, uniparental chromosome elimination results in the development of a haploid
embryo. The mitosis-dependent impaired uniparental chromosome segregation is driven by either (i ) centromere inactivity or (ii ) the
compromised release of sister chromatid cohesion. Cell cycle asynchrony (for example, caused by genetic differences) may, in an
unstable hybrid, interfere with the timely loading of CENH3 onto the paternal nucleosomes. Whether impaired sister chromatid
cohesion and centromere inactivity act together remains unknown. (iii ) The mitosis-independent process of chromosome elimination
results in the budding of parental chromatin during interphase. (iv) As a result, centromere-inactive micronuclei arise. Micronucleated
chromatin undergoes heterochromatinization and transcription inactivation. Embryonic cells harbor a haploid nucleus once the
micronuclei have fully degenerated.
malfunction of any of which can produce a nonfunctional centromere (for a review, see 66). The
only plant proteins so far implicated in the establishment and maintenance of CENH3 chromatin
are the A. thaliana gene products Mis12 (81), KNL2 (55), and GIP (3). Because the correct timing
of CENH3 deposition appears to be critical to ensure normal cell division, cell cycle asynchrony—
arising, for example, from a difference in cell cycle time between a hybrid’s parental species—may
compromise CENH3 loading onto the nucleosomes of one of the parents. A feature of many un-
stable hybrids is the variation in the degree of chromosomal condensation observed between the
parental genomes. The possibility that other factors are responsible for assembly failure also can-
not be excluded. Given that the elimination process is temperature labile, it may well be relevant
that nucleosome composition—specifically, an altered deposition of histone variants—is similarly
temperature dependent (48). Such temperature-mediated changes in centromeric nucleosome as-
sembly can therefore explain the temperature effect on the uniparental chromosome elimination
process. Whether the chaperones required for CENH3 loading are temperature sensitive is un-
known. In hybrids between several cereals other than barley (2x, 4x, and 6x wheat; triticale; rye;
and oat) and pearl millet, the process of uniparental chromosome elimination has been ascribed
to an ineffective release of sister chromatids (40), but whether this process acts together with
centromere inactivity remains unclear.
In addition to chromosome loss during mitosis, elimination can also take place during inter-
phase. Chromatin-containing structures resembling micronuclei in both shape and size have been
observed attached to interphase nuclei (25, 26). These micronuclei appear to have been expelled
from the interphase nucleus, although it is also possible that they are in the process of fusion with,
rather than fission from, the main nucleus (the reverse of elimination). Selective elimination of
the male gamete’s chromosomes achieved via the formation of nuclear extrusions is reminiscent
of the double minutes seen in certain mammalian tumor cells (91), raising the intriguing pos-
sibility of an evolutionarily conserved process that distinguishes host from foreign or damaged
chromatin/DNA and targets the latter for removal. The extent to which this type of mechanism
comes into play in other interspecific cross combinations is unknown.
THE ELIMINATION OF A MICRONUCLEATED GENOME

The majority of micronuclei arising from lagging anaphase chromosomes contain only a small
number of chromosomes, although large micronuclei with up to seven chromosomes have occa-
sionally been observed (26). Ultrastructural analysis has shown that micronuclei are surrounded
by a double membrane pierced with pores, mirroring the structure of a normal nucleus. However,
micronucleated chromatin differs from the usual type in several ways: It displays a lower number
of anti–RNA polymerase II signals and an increased level of the heterochromatin mark histone
H3 dimethylated at lysine 9, and the DNA is clearly fragmented; all of these features indicate
a reduced level of transcriptional activity. Because the centromeres of micronucleated chromo-
somes are inactive, segregation does not occur (26, 79). Their subsequent elimination seems to
result from their distinct chromatin topology, which causes endonuclease-mediated fragmenta-
tion. Posttranslational histone modifications could help promote and direct the drastic changes
to the DNA’s integrity and chromatin compaction that are imposed on the eliminated genome. A
comparable process has been described for chromosomes eliminated in sciarid flies (28) and zebra
finches (Taeniopygia guttata) (27).
CENH3 BEHAVIOR IN STABLE HYBRIDS

In stable hybrids, the CENH3 produced by one parent can support the functionality of the other
parent chromosomes’ centromeres, despite differences in the two parents’ centromere sequences.
This phenomenon has allowed the creation of a substantial number of wide hybrids and an even
larger number of derived lines containing incomplete donor parent genomes. Some pairs of
rather remotely related species (a prime example is oat and maize) can be successfully crossed
F1 hybrid:
a cross between two to produce a fertile partial hybrid. In the oat × maize combination, oat CENH3 must compen-
nonidentical sate for the absence of functional maize CENH3 because the maize CENH3 gene is silenced
genotypes; when the (41). Similarly, A. thaliana CENH3 is detectable at the centromere of each chromosome of the
parents are both A. thaliana × Arabidopsis arenosa hybrid (89). Cross-species incorporation of CENH3 also occurs
homozygous, the
in stable H. vulgare × H. bulbosum hybrids. In a wheat-barley 1H + 6H addition line, despite the
hybrid usually
ountperforms both transcription of the genes encoding the barley α- and βCENH3 proteins, only the presence of the
parents owing to αCENH3 could be verified (79). This outcome likely reflects the closer homology of the wheat
heterosis CENH3s with the barley αCENH3 than with the barley βCENH3, which allows the former
to be readily loaded onto the centromeres of both wheat and barley chromosomes. Similarly, in
the oat × pearl millet hybrid, despite the expression of both parental CENH3 genes, only the oat
product is loaded (39). The loop 1 regions of the oat and pearl millet CENH3 sequence exhibit an
exceptional level of similarity. An analysis of transgenic materials involving CENH3 sequences has
confirmed that cross-species CENH3 incorporation can occur (listed in 57). Unanswered ques-
tions include how many different CENH3 variants are loaded onto the centromeres of polyploid
species, to what degree crossability between species depends on the ability of centromeres to in-
corporate the different parental CENH3 variants, and whether each CENH3 variant employs its
own set of assembly factors in a hybrid.
GENERATION OF HAPLOIDS USING CROSSES WITH INTRASPECIFIC

HAPLOID INDUCER LINES
Intraspecific haploid inducers have become the foundation of modern DH technology in maize
(reviewed in 7, 24, 70). Through selection, the frequency of haploids in the inducer line Stock 6
has increased from 1–2% (9) to 8–10% (97). Haploid progeny can be recognized by exploiting the
R1-nj anthocyanin marker system (65) or by measuring oil content (60). The induction mechanism
is not yet understood. Sarkar & Coe (80) have suggested that a haploid embryo develops from
an unfertilized egg cell, resulting in a chimera in which the embryo proper is maternal but the
suspensor cells are paternal, but a more likely explanation is that the inducer’s chromosomes are
eliminated after fertilization (94, 99).
In an attempt to test these conflicting hypotheses, Zhao et al. (100) generated inducer lines
containing either supernumerary B chromosomes or a CENH3-YFP transgene in order to recog-
nize the pollinator chromosomes in the hybrid. Pollinator chromosome fragments were detected
in F1 haploids at a low frequency, providing direct evidence for selective chromosome elimination
during haploidization. Most of the inducer chromosomes were eliminated within the first week
after fertilization, a rate comparable to that occurring in unstable H. vulgare × H. bulbosum em-
bryos (25, 86). Chromosome elimination in the maize endosperm is both partial and less rapid,
leading to the formation of kernels that have a normal endosperm but a haploid embryo (100). Xu
et al. (97) were able to confirm that a double fertilization occurs during haploid induction, further
implying that chromosome elimination is the basis of haploid induction in maize. Some chromo-
somes are nevertheless able to escape elimination, with the B recovered most often, presumably
because disomic haploids involving A chromosomes are lethal or highly defective, but the nearly
inert B as an extra chromosome is without consequence (100).
A major locus that is responsible for haploid induction and expressed in the male gamete has
been mapped to maize chromosome 1 (2). Thus, maternal haploid induction is unlikely to be due
to a CENH3 mutation, because this gene maps to chromosome 6, and no homologs are known
within the relevant bin on chromosome 1 (71). Furthermore, no difference between the CENH3
gene of the inducer and noninducer lines has been identified with respect to either the gene’s
coding sequence or its transcription profile (100). Rather, the proposition is that the segregation
distortion 1 (sed1) gene imposes an epigenetic, dosage-dependent chromosome modification (97).
The assumption is that the expression of sed1 differs between the pollen of plants homozygous
for the sed1 haplotype. Consequently, the nature of the chromosome modification induced by the
action of the sed1 product can differ from pollen grain to pollen grain. Some modifications to
the male gamete’s chromosomes can result in either kernel abortion or haploid formation. Both
gametophytic and sporophytic selection are involved in haploid induction, and these two mech-
anisms together produce major segregation distortion with respect to sed1. A major quantitative
trait locus accounts for up to 66% of the variance in haploid induction rate (71), but several other
loci likely act as enhancers for its function (14). Fine-scale mapping has defined a 243-kb segment
as the site of the major locus (14).
An additional haploid-inducing gene in maize is represented by indeterminate gametophyte (ig),

which maps to chromosome 3 (45, 46) and encodes a LATERAL ORGAN BOUNDARIES (LOB)
domain protein. Loss of function results in the formation of haploids of both maternal and paternal
origin, raising the possibility of exploiting this gene to produce paternal haploids harboring a
maternal cytoplasm (46). In this case, it is not uniparental chromosome elimination that causes
haploid formation; rather, the gene restricts the proliferative phase of the female gametophyte’s
development. The mutant’s female gametophytes experience a prolonged phase of coenocytic
divisions, which results in a variety of embryo sac abnormalities, including supernumerary egg cells,
polar nuclei, and synergids (18). Note that other proteins containing a LOB domain act to define
organ boundaries and are involved in many aspects of plant development (for a review, see 58).
In barley, the haploidy initiator gene hap prevents the fertilization of the egg cell but does not
affect the polar cell or interfere with endosperm development (30, 64). Because of the latter, there
is no need for a tissue culture intervention to rescue the embryo. Depending on both genotype
and environment, the progeny of hap homozygotes can include up to 40% haploids, along with
several spontaneous DHs. Unfortunately, the haploid frequency from a hap heterozygote is low
(0.1–10%) (31), which minimizes the utility of this mutant for plant breeding.
GENERATION OF HAPLOIDS USING TARGETED

CENTROMERE MANIPULATION
The work of Ravi & Chan (73), which showed that manipulation of CENH3 mimics some of the
outcomes of unstable inter- or intraspecific hybrids, represents a breakthrough in the induction
of haploids. Chromosomes of the transgenic parent are eliminated during early embryogenesis
in either crossing direction. Besides haploidization, this approach can facilitate the transfer of
a given nucleotype into an alternative cytoplasm. Centromere-mediated haploidization also has
other potential applications: as a tool to accelerate the pyramiding of multiple mutants, as a Targeting induced
local lesions in
forward mutagenesis screen, for downsizing a tetraploid to a lower ploidy level, and for generating
genomes
homozygotes for gametophyte-lethal mutations (74). Haploids can be exploited to rapidly generate (TILLING):
mapping populations (22, 84), chromosome substitution lines, and parents for reverse breeding a reverse genetics
(95, 96) (see sidebar Reverse Breeding) and to engineer apomixes (59). method designed to
A combination of uniparental chromosome elimination and MiMe (see sidebar MiMe: Mitosis identify mutations
within a specific DNA
Instead of Meiosis) has been proposed as a means to produce clonal seeds. Ravi & Chan (73)
sequence following a
constructed a chimeric histone H3.3/CENH3 protein by fusing the N-terminal tail of a canonical random mutagenesis
A. thaliana H3.3 histone with a green fluorescent protein (GFP) reporter, then substituted this treatment
construct for the CENH3 tail. The resulting “GFP-tailswap” protein complements the lethal
phenotype of a cenh3 null mutant generated using the targeting induced local lesions in genomes
REVERSE BREEDING
Fixation of a heterozygote is impossible without apomixis or vegetative reproduction. Reverse breeding is designed
to generate complementary homozygotes for use as parents of an F1 hybrid. The method is based on the suppression
of meiotic recombination, which is achieved by knocking down genes essential for crossover formation (e.g., ASY1,
DMC1, or SPO11) while leaving chromosome structure intact. The haploid sporocytes of such engineered plants
are regenerated into DH plants, among which genetically complementary and completely homozygous individuals
can be selected. Intercrossing these plants allows the essentially unlimited replication of an elite heterozygote.
(TILLING) method. Between 25% and 45% of the progeny of the transgenic CENH3−/− haploid
inducer line are haploid when the line is the maternal parent, but the rate drops to 5% when the
line is the paternal parent. Self-fertility is lower owing to partial male sterility. CENH3+/− lines
are fertile, and the null allele is transmissible.
In a further development, the efficient selection of haploid seeds was facilitated by introduc-
ing GFP under the control of the seed storage protein 2S3 promoter to produce the so-called
SeedGFP–haploid inducer (74). Haploid seeds that lack GFP expression in their embryo while
their endosperm still displays some fluorescence are thought to reflect the partial loss of the mu-
tant parent’s chromosomes in the endosperm. The nonproduction of haploid endosperm–haploid
embryo seed is likely dependent on the need to maintain a 2:1 ratio of parental genome dosage in
the endosperm.
Because the GFP-tailswap-derived chromosomes are not fully eliminated, several aneuploid
progeny are generated. Sequence analysis has revealed that the process also generates truncated
and shattered chromosomes (90). Mutating the gene encoding the DNA ligase 4 enzyme required
for nonhomologous end joining increases the recovery rate of haploids (90), perhaps reflecting the
presence of unrepaired double-strand breaks, as occurs in unstable somatic mouse-human hybrid
cells (93). A plausible scenario is that mis-segregated chromosomes are channeled into a degrada-
tive pathway initiated by DNA fragmentation. Occasionally, such chromosomes may be rescued
through a nonhomologous end joining pathway, resulting in restructured chromosomes (90). The
restructuring of chromosomes undergoing elimination also occurs in interspecific hybrids (26, 40).
The generation of copy-number variants and chimeric genes, along with their occasional meiotic
transmission, suggests that chromosomal restructuring caused by a centromere-based mechanism
of instability could contribute to the establishment of heritable genetic variation (90). Such a
mechanism may even be exploitable for the de novo formation of B chromosomes, given that B
chromosomes are often a mosaic of standard chromosome fragments (for a review, see 34).
MiMe: MITOSIS INSTEAD OF MEIOSIS
A key element of apomixis is apomeiosis, a deregulated form of meiosis that results in a mitosis-like division.
Combining a loss-of-function mutant of the Arabidopsis thaliana gene OSD1, which controls entry into the second
meiotic division, with two other mutants affecting key meiotic processes (spo11-1 and rec8) produced the so-called
MiMe genotype, in which meiosis was completely replaced by mitosis. The plants produced functional, genetically
identical diploid gametes. A drawback is that the ploidy level would be expected to increase at each generation
because of the absence of the meiotic reduction division. However, this obstacle can be overcome by exploiting
uniparental chromosome elimination, an approach that has been used to generate clonal seed in A. thaliana.
A somewhat less efficient haploid inducer effect is displayed by A. thaliana cenh3 null mutants
harboring a heterologous CENH3 gene not tagged with GFP (57). These plants are self-fertile, but
their hybrid with a wild-type plant suffers segregation errors, notably involving the chromosomes
CENH3
inherited from the mutant. Although Lepidium oleraceum is more closely related to A. thaliana than centromere-
is Brassica napus, replacing the A. thaliana CENH3 copy with that from L. oleraceum had a greater targeting domain
effect on A. thaliana centromere function, as inferred from a higher frequency of induced haploids. (CATD): a domain
To better define CENH3 functional constraints, Kuppu et al. (50) complemented a CENH3 defined by loop 1 and
the α2 helix of
null allele in A. thaliana with a variety of mutant alleles, each inducing a single amino acid change in
CENH3 that is
conserved residues of the histone fold domain. Many of these transgenic missense lines displayed required for the
wild-type growth and fertility on self-pollination but exhibited frequent postzygotic death and protein to load onto
uniparental inheritance when crossed with wild-type plants. To demonstrate this approach, the the centromere
authors performed an in silico search for previously identified point mutations in CENH3 and
identified a nontransgenic A. thaliana line carrying an A86V substitution within the histone fold
domain. This line, although fully fertile on self-pollination, produced uniparental haploids when
crossed with the wild type.

In parallel, the effect on centromere function of small mutations in the endogenous CENH3
sequence has been tested in a TILLING experiment conducted in barley (43). An amino acid
exchange at a site within the centromere-targeting domain (CATD) of βCENH3 is associated
with a reduced centromere loading of the protein. The same mutation results in reduced loading
of CENH3 in transgenic Arabidopsis and sugar beet. In A. thaliana, haploids were obtained after
crossing a complemented cenh3 null mutant containing the same mutation of the conserved amino
acid (CENH3 L130F) with wild-type A. thaliana. By contrast, in a noncompeting situation in
which the centromeres possess either only mutated or only wild-type CENH3 during early em-
bryogenesis, no uniparental chromosome elimination occurred. The cross-species conservation
of critical CENH3 sites (43, 50) suggests an interesting strategy for inducing haploidy in a range
of crop species.
Figure 2 illustrates possible mechanisms for the operation of CENH3-based elimination.
Chromosome mis-segregation in the F1 zygote likely results from competition between wild-
type and defective centromeres. Haploid inducer–derived egg cells are loaded either with less
CENH3 or with a CENH3-transgeneration-required signature (compared with the wild type)
(43, 50). Based on the behavior of a wild-type CENH3-GFP fusion reporter gene in A. thaliana,
the nuclei of sperm—but not those in the egg cell—are marked with CENH3 (38). Nonetheless,
it is still possible that residual maternal CENH3, which is able to impose centromeric imprinting,
is transmitted to the progeny. Alternatively, the loss of egg cell–loaded CENH3 may be species
specific, as the presence of CENH3 has been demonstrated in the egg cell of oat (39). Within a few
hours of fertilization, paternal wild-type CENH3 is actively removed from the A. thaliana zygote
nucleus, and centromeric reloading of CENH3 occurs at the 16-nucleus stage of the immature
endosperm (38). In embryos undergoing haploidization, centromeric reloading of the maternal
chromosomes is either impaired or delayed, causing chromosomes to lag owing to the loss of
centromere function at anaphase. Subsequently, micronucleated haploid inducer chromosomes
degrade, resulting in the formation of a haploid embryo, as demonstrated in unstable interspecific
hybrid embryos (26). Competition between strong and weak centromeres is also employed by some
Mendelian lawbreakers to subvert the process of chromosome segregation in their own favor.
In mice, unequal CENH3 loading on the centromere during female meiosis has been directly
associated with the meiotic drive displayed by Robertsonian fusion chromosomes (8). Stronger
centromeres, manifested by increased kinetochore protein levels, exhibit an altered interaction
with the microtubule spindles and are preferentially retained in the egg.
Unstable intraspecific cross Stable intraspecific cross

CENH3 CENH3 CENH3 CENH3
1 1
parents
Diploid
parents
Diploid
Somatic
Somatic
cells
cells
*
*
2 2
Egg cell Sperm cell Egg cell Sperm cell
Gametes
Gametes
* × ×
3 3
zygote
zygote
Early
Early
*
i
4 4
Embryogenesis
Embryogenesis
ii
iii x iv
5 5
Haploid Aneuploid Diploid
* Haploid inducer CENH3-positive centromeres Micronucleus derived from a lagging chromosome

because of centromere inactivity
Wild-type CENH3-positive centromeres Micronucleus reintegrating into the nucleus
* Centromeres with residual amounts of x Micronucleus undergoing degeneration

haploid inducer CENH3
Maternal cytoplasm
Centromeres with residual amounts of CENH3
Paternal cytoplasm
Figure 2
A model for the uniparental chromosome elimination process that occurs in haploid inducer CENH3 × wild-type CENH3 intraspecific
hybrid embryos (unstable intraspecific cross) (left) compared with wild-type embryo formation (stable intraspecific cross) (right).
() The somatic cell chromosome centromeres in a homozygous haploid inducer contain less CENH3 than do wild-type centromeres.
() Haploid inducer–derived egg cell chromosomes accumulate either less CENH3 or less of an unknown CENH3-transgeneration-
required signature. () After fertilization, wild-type CENH3 is actively removed from the paternal chromosomes in the zygote’s
nucleus. () Centromeric reloading of wild-type CENH3 in the zygote occurs. () In an embryo undergoing haploidization,
centromeric reloading of the maternal chromosomes is either impaired or delayed, resulting in (i ) lagging chromosomes and (ii ) the
formation of micronuclei. (iii ) Micronuclei degrade, as they also do in unstable wide hybrids, and a haploid embryo develops. (iv) DNA
damage and its nonhomologous end joining–facilitated repair may occur. The fusion of a micronucleus with a full nucleus can generate
aneuploidy if the centromere activity of the micronucleated chromosome is maintained.
CHROMOSOME-TYPE-SPECIFIC ELIMINATION AS PART OF CELL

DIFFERENTIATION AND SEX DETERMINATION
In addition to CENH3-based chromosome elimination, other ways of eliminating a parental
genome or specific chromosomes can form a part of normal cell differentiation and sex determina-
tion. Programmed DNA elimination occurs in certain unicellular ciliates and various metazoans. In
most cases, the process is associated with either the differentiation of somatic cells or sex determi-
nation. Two types of programmed DNA elimination have been described: chromatin diminution
and chromosome elimination. In the former, the chromosomes fracture and some chromosomal
segments are lost (in, e.g., ciliates and copepods), whereas in the latter, entire chromosomes are
lost (in, e.g., some insects and finches). Given its wide phylogenetic distribution, programmed
DNA elimination likely evolved independently several times. The frequency of programmed
DNA elimination has probably been underestimated, because generally only a single tissue type
is analyzed. A variety of hypotheses have been formulated about the significance of programmed
DNA elimination; the proposed explanations include sex determination, dosage compensation,
gene silencing, germline development and meiosis, and germline and soma differentiation (for
reviews, see 47, 92). The key question is how the chromosomes are selectively lost.
SEX DETERMINATION VIA CHROMOSOME ELIMINATION

The association between sex determination and chromosome elimination was first described in
the dipteran genus Sciara, which exhibits a complex pattern of chromosome elimination and sex
determination (62). In Sciara coprophila, sex is not determined until the early stage of embryogenesis.
One or both of the paternal X chromosomes are eliminated from somatic cells to form the female
or male individual, respectively. At a later stage of the embryo’s development, a single paternal
X chromosome is eliminated from the germ cells of both sexes. At male meiosis I, the entire
set of paternal chromosomes is eliminated, and during meiosis II, the maternal X chromosome
undergoes nondisjunction and creates sperm bearing two X chromosomes. Crouse (10) suggested
that eliminated chromosomes are imprinted. In S. coprophila, a cis-acting element regulates both the
elimination of the paternal X chromosome during embryo development and the nondisjunction
of the maternal X chromosome during male meiosis. The translocation of the nondisjunction
control region to an autosome results in the nondisjunction of the recipient chromosome and the
normal segregation of the X chromosome lacking the controlling region. Neither the sequence
composition nor the action of the nondisjunction control region has been determined. However,
nondisjunction in Sciara resembles the meiotic drive of rye B chromosomes, which may result from
the in trans regulation exerted by noncoding RNA over the nondisjunction control region (6).
Because delayed sister chromatid separation at anaphase is correlated with the hyperphospho-
rylation of histone H3 on the X chromosome, Escriba & Goday (17) suggested that a cis-acting
locus acts to inhibit H3 dephosphorylation in the paternal X chromosomes, marking them for
elimination. In the one-factor model proposed by de Saint Phalle & Sullivan (13), the imprinted
paternal X chromosome interacts with a maternal factor to induce the failure of sister chromatid
separation, whereas an alternative two-factor model suggests that an additional maternal factor
regulates the amount of chromosomal factors interacting with the X chromosome to be eliminated
(78). Sex chromosomes are also eliminated in certain vertebrates, such as the marsupial species
Perameles nasuta, Isoodon obesulus, and Isoodon macrourus (32).
Sex change effected by chromosome elimination, operating through a haplodiploid sex deter-
mination mechanism, has been demonstrated in the parasitoid wasp Nasonia vitripennis; offspring
that lack the paternal genome are haploid and male, whereas those that retain it are diploid and
female (87). Two distinct haploidization mechanisms can act during early embryogenesis. In the
first, chromosome elimination is initiated by a supernumerary, paternal sex ratio (PSR) chromo-
some. Fertilization involving a sperm carrying the PSR chromosome results in the elimination of
the full male gamete parental chromosome complement except for the PSR chromosome. The
PSR chromosome likely imprints the paternal chromosomes via an alteration to the histone H3
phosphorylation profile in combination with its effect on the loading of the condensin complex
(87). Both transcripts encoding putative proteins and long noncoding RNA are generated in PSR-
harboring individuals (1). The second haploidization mechanism is exemplified by bacteria from
the genus Wolbachia, in which the cytoplasm disrupts paternal chromosome condensation (76).
ELIMINATION OF GERMLINE-RESTRICTED CHROMOSOMES

In the zebra finch, a germline-restricted chromosome (GRC) is frequently observed in both sexes
(68). Elimination of this chromosome occurs during early embryogenesis and in male meiosis.
The GRC is present as a single copy in the spermatocyte, but the female germline has two
copies, which synapse normally. Whereas the male GRC is highly condensed and appears to be
heterochromatic, the female one is euchromatic. Meiotic GRCs undergoing elimination become
enriched in heterochromatin protein 1, and histone H3 on this chromosome is methylated at
lysine 9. Furthermore, histone modifications (histone H3 methylated at lysine 4 and histone H4
acetylated) diagnostic for euchromatin become depleted. In GRCs during the elimination process,
but not during meiotic prophase, the phosphorylated form of histone H3 at serine 10, a marker
for condensed chromosomes in vertebrates, becomes prominent (27). Because the GRC lacks
the inner centromere protein INCENP during male meiosis, its post–metaphase I elimination is
thought to be linked to centromere inactivation. Subsequently, the GRC forms a micronucleus
containing fragmented DNA (82).
The elimination of supernumerary B chromosomes is tissue specific across a diverse range
of species (for a review, see 42). Typically, B chromosomes are not propagated in the root but
persist in the aerial part of the plant. Examples of this phenomenon have also been documented
in animals, such as the ant Leptothorax spinosior, in which B chromosomes are restricted to germ
cells of males and are rarely found at all in females, either in germ cells or in somatic cells (37).
CONCLUDING REMARKS AND OUTLOOK

The exploitation of haploidization by targeted centromere manipulation for the purpose of crop
improvement requires the elaboration of a cenh3 null mutant in combination with a modified
CENH3 variant that reduces the effectiveness of the centromere compared with the wild type.
Given recent advances in targeted mutagenesis (72), any plant that can be successfully transformed
can be rendered defective in CENH3. Alternatively, but more laboriously, a TILLING approach
could conceivably permit the identification of individuals with partially impaired centromere func-
tion; note, however, that a large genetic screen for haploid inducers in A. thaliana has failed to
identify any suitable mutants (69). As the female-partner-based CENH3 haploidization process
is more efficient, a rational next step would be to determine its genetic control. Experiments in
Drosophila melanogaster have provided indirect evidence that CENH3 manipulation could result in
haploidization in species other than A. thaliana. In these experiments, Raychaudhuri et al. (75) used
targeted protein degeneration to substantially deplete levels of Cid (the Drosophila CENH3 equiv-
alent) in the sperm; this led to a failure of the paternal centromeres to integrate into the gonomeric
spindle of the first mitosis and the subsequent formation of gynogenetic haploid embryos.
The combination of in vivo haploidization and designer minichromosomes (see sidebar En-
gineered Minichromosomes) could represent, at least in principle, an attractive strategy for
ENGINEERED MINICHROMOSOMES
Conventional transformation vectors have a limited capacity to harbor multiple transgenes. It is not simple to control
the number of transgene copies inserted, nor is it possible to avoid mutagenesis at their site of insertion into the host
genome. Minichromosomes, constructed by reducing the length of an extant chromosome via telomere-mediated
truncation, in principle avoid all of these problems. They provide the means to stack a large number of transgenes.
Because a minichromosome replicates autonomously, it can be incorporated as an independent chromosome in the
host genome, rather than needing to be inserted within an existing chromosome.
transferring blocks of transgenes between lines within a species (23). The substantial epigenetic
component underlying the centromere structure complicates the in vivo production of synthetic
plant chromosomes based on cloned building blocks. The truncation of existing host chromosomes
to use endogenous centromeres could be a possible way to circumvent the need to generate de
novo centromeres (98). As telomeric repeats are highly conserved in the plant kingdom, telomere-
mediated truncation should be achievable in most plant species (for a review, see 61). Because
haploid inducers of various types often contribute a chromosome to haploids, as noted above,
incorporation of engineered minichromosomes into these lines might allow their transfer to hap-
loids of target lines that could be doubled to bypass a lengthy transgene introgression scheme (23).
The future of precise plant breeding looks promising.
SUMMARY POINTS
1. A uniparental chromosome segregation defect during early embryogenesis underlies the
process of in vivo haploidization in unstable inter- and intraspecific hybrids.
2. Changes to CENH3 (e.g., replacement of the N-terminal tail of CENH3 with the tail
of conventional histone H3, complementation with CENH3s of distant relatives, or a
point mutation) create a haploid inducer phenotype in Arabidopsis thaliana.
3. The high degree of evolutionary conservation of CENH3 offers a promising strategy for
inducing haploidization in a wide range of crop species.
4. In the majority of unstable interspecific hybrids, the chromosome set of the male ga-
metophyte is eliminated. By contrast, haploidization is more efficient in intraspecific
A. thaliana–based wide hybrids when the CENH3-based inducer is used as the female
parent of the cross.
5. Chromosome elimination exists as part of normal development during cell differentiation
and sex determination in various species.
6. Other means of eliminating a parental genome or specific chromosomes exist apart from
the centromere-based mechanism.
FUTURE ISSUES
1. The elaboration of relevant protocols is required before targeted CENH3 manipulation
can be applied to crops to induce haploidization.
2. An analysis of the process of haploidization in unstable interspecific hybrids and in haploid

inducers in maize should identify the mechanisms that underlie haploidization in addition
to the one based on centromeres.
3. Removing the need to deploy embryo rescue as a result of impaired endosperm develop-
ment would be advantageous. This could be achieved by restricting uniparental genome
elimination to the embryo.
4. The capacity to induce synthetic apomixis would allow for seed multiplication of nonho-
mozygous elite materials. Achieving this by combining MiMe (in which mitosis replaces
meiosis) with uniparental chromosome elimination will require improvements in the
penetrance of the haploid inducer line.
5. Besides gaining a better understanding of the interaction between parental genomes in
an unstable wide cross, research may deliver improved methods of generating stable
wide-cross combinations.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
We apologize to authors whose research could not be discussed here owing to space restrictions.
This research was supported by the German Federal Ministry of Education and Research (Plant
2030 Project HAPLOIDS FKZ 0315965). We appreciate many helpful discussions with various
members of our laboratory.
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PP67CH10-Houben ARI 7 January 2016 14:17

Review in Advance first posted online

still occur before final publication

Takayoshi Ishii, Raheleh Karimi-Ashtiyani,

and Andreas Houben

Annu. Rev. Plant Biol. 2016. 67:10.1–10.18 Keywords

This article’s doi: Abstract

SEX DETERMINATION VIA CHROMOSOME ELIMINATION . . . . . . . . . . . . . . .10.11

CONCLUDING REMARKS AND OUTLOOK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.12

10.2 Ishii · Karimi-Ashtiyani · Houben

www.annualreviews.org • Haploidization via Chromosome Elimination 10.3

Unstable interspecific cross Stable interspecific cross

Female CENH3-positive centromeres Micronucleus derived from lagging chromosome

10.4 Ishii · Karimi-Ashtiyani · Houben

THE ELIMINATION OF A MICRONUCLEATED GENOME

CENH3 BEHAVIOR IN STABLE HYBRIDS

www.annualreviews.org • Haploidization via Chromosome Elimination 10.5

GENERATION OF HAPLOIDS USING CROSSES WITH INTRASPECIFIC

10.6 Ishii · Karimi-Ashtiyani · Houben

An additional haploid-inducing gene in maize is represented by indeterminate gametophyte (ig),

GENERATION OF HAPLOIDS USING TARGETED

www.annualreviews.org • Haploidization via Chromosome Elimination 10.7

MiMe: MITOSIS INSTEAD OF MEIOSIS

10.8 Ishii · Karimi-Ashtiyani · Houben

crossed with the wild type.

www.annualreviews.org • Haploidization via Chromosome Elimination 10.9

Unstable intraspecific cross Stable intraspecific cross

Haploid Aneuploid Diploid

* Haploid inducer CENH3-positive centromeres Micronucleus derived from a lagging chromosome

* Centromeres with residual amounts of x Micronucleus undergoing degeneration

10.10 Ishii · Karimi-Ashtiyani · Houben

CHROMOSOME-TYPE-SPECIFIC ELIMINATION AS PART OF CELL

SEX DETERMINATION VIA CHROMOSOME ELIMINATION

www.annualreviews.org • Haploidization via Chromosome Elimination 10.11

ELIMINATION OF GERMLINE-RESTRICTED CHROMOSOMES

CONCLUDING REMARKS AND OUTLOOK

10.12 Ishii · Karimi-Ashtiyani · Houben

www.annualreviews.org • Haploidization via Chromosome Elimination 10.13

2. An analysis of the process of haploidization in unstable interspecific hybrids and in haploid

10.14 Ishii · Karimi-Ashtiyani · Houben

at early cleavages in sciarid flies. J. Cell Sci. 126:3214–22

for embryo sac and leaf development. Plant Cell 19:46–62

www.annualreviews.org • Haploidization via Chromosome Elimination 10.15

functional adaptation in the genetic background of oat. Plant Cell 16:571–81

10.16 Ishii · Karimi-Ashtiyani · Houben

www.annualreviews.org • Haploidization via Chromosome Elimination 10.17

10.18 Ishii · Karimi-Ashtiyani · Houben

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