Research

Asexual reproduction in a close relative of Arabidopsis:

Blackwell Publishing Ltd

a genetic investigation of apomixis in Boechera

(Brassicaceae)
M. Eric Schranz1,*, Laksana Kantama2, Hans de Jong2 and Thomas Mitchell-Olds1
1
Department of Genetics and Evolution, Max Planck Institute for Chemical Ecology, Hans Knoll Strasse 6, D-07745 Jena, Germany; 2Laboratories of
Biochemistry and Genetics, Wageningen University, Arboretumlaan 4, NL-6703 BD Wageningen, the Netherlands; *Present address: Department of Biology,
Box 91000, Duke University, Durham, NC 27708, USA

Summary

Author for correspondence: • Understanding apomixis (asexual reproduction through seeds) is of great interest
M. Eric Schranz to both plant breeders and evolutionary biologists. The genus Boechera is an excellent
Tel: +1 (919) 613 8181 system for studying apomixis because of its close relationship to Arabidopsis, the
Fax: +1 (919) 613 8177
Email: eric.schranz@duke.edu
occurrence of apomixis at the diploid level, and its potentially simple inheritance by
transmission of a heterochromatic (Het) chromosome.
Received: 3 March 2006
• Diploid sexual Boechera stricta and diploid apomictic Boechera divaricarpa
Accepted: 24 March 2006
(carrying a Het chromosome) were crossed. Flow cytometry, karyotype analysis,
genomic in situ hybridization, pollen staining and seed-production measurements
were used to analyse the parents and resulting F1, F2 and selected F3 and test-cross
(TC) generations.
• The F1 plant was a low-fertility triploid that produced a swarm of aneuploid and
polyploid F2 progeny. Two of the F2 plants were fertile near-tetraploids, and analysis of
their F3 and TC progeny revealed that they were sexual and genomically stabilized.
• The apomictic phenotype was not transmitted by genetic crossing as a single
dominant locus on the Het chromosome, suggesting a complex genetic control of
apomixis that has implications for future genetic and evolutionary analyses in this group.

Key words: apomixis, Boechera (Brassicaceae), interspecific hybridization, polyploidy,

genome painting, heterochromatic chromosome.

New Phytologist (2006) 171: 425–438

© The Authors (2006). Journal compilation © New Phytologist (2006)

doi: 10.1111/j.1469-8137.2006.01765.x

directly derived from normal sexual pathways (Nogler, 1984a;

Introduction
The study of apomixis (asexual reproduction through seeds)
is of great interest for both practical and theoretical reasons.
The harnessing of apomixis for agricultural purposes could be
of great economic and humanitarian benefit, because it would
enable the propagation of hybrid genotypes indefinitely
(Spillane et al., 2004). For evolutionary biologists, apomictic
systems allow for testing of hypotheses of the persistence of sexual
reproduction despite its twofold cost (Charlesworth, 1990;
West et al., 1999). Although biologists have long grappled with
apomictic mechanisms, their control is still relatively poorly
understood at the genetic and molecular levels (Bicknell &
Koltunow, 2004). It has been hypothesized that apomixis is
Holsinger, 2000; Koltunow & Grossniklaus, 2003). This
bypassing of normal sexuality may have resulted from gene-
expression changes during polyploidization and/or hybridization,
as most apomictic species are allopolyploids (Carman, 1997).
A number of model species have been well developed for the
study of apomixis, each with their own advantages and disad-
vantages (Bicknell & Koltunow, 2004). Genetic studies with
these systems suggest that the trait is under the control of either
two or several genes (van Dijk et al., 1999; Noyes & Rieseberg,
2000; Albertini et al., 2001; Matzk et al., 2005), or by a single
dominant locus (Leblanc et al., 1995; Bicknell et al., 2000),
which can be located on supernumerary or hemizygous chromatin
(Ozias-Akins et al., 1998; Roche et al., 2001; Sharbel et al., 2004).

www.newphytologist.org 425
426 Research
In comparison with other apomictic complexes, apomictic
reproduction in Boechera has several factors making it more
akin to normal sexual reproduction. These factors combine to
make Boechera a compelling and tractable system for genetic
studies of the control of apomixis. Perhaps the most promis-
ing and rare characteristic of Boechera is that apomixis can
occur at the diploid level (Böcher, 1951, 1969; Sharbel &
Mitchell-Olds, 2001). Evidence of diploid apomictic Boechera
lines has been gathered through cytological investigations
(Böcher, 1951; Naumova et al., 2001); the occurrence of unre-
duced gametes and heterozygous genotypes (DobeS et al., 2004b;
Sharbel et al., 2004, 2005); and the persistence of heterozygosity
in progeny (Roy, 1995; Schranz et al., 2005). Additionally, these
diploid lines often contain heterochromatic (Het) and/or
supernumerary chromosomes that could be responsible for
the apomictic phenotype (Böcher, 1954; Sharbel et al., 2004;
Kantama, 2005; Sharbel et al., 2005). Another major
advantage is the close relationship of Boechera to the model
plant Arabidopsis thaliana (Koch et al., 2001). Boechera is
the only documented case of natural apomixis in the Brassica
family (Brassicaceae). In addition to A. thaliana having its
complete genome sequenced, there is also an unrivalled
understanding of normal sexual reproduction and a variety of
apomixis-like mutations that have been identified (Koltunow
& Grossniklaus, 2003), which should provide an excellent
framework for comparison.
Several aspects of the Boechera breeding system simplify genetic
and molecular investigations, compared with other apomictic
lineages. First, apomictic Boechera accessions have probably
evolved from self-compatible and highly self-fertilizing sexual
types (Roy, 1995), whereas most other apomictic groups are
derived from self-incompatible and out-crossing taxa (Asker
& Jerling, 1992). Second, as in normal sexual taxa, the embryo
is derived from the megaspore mother cell (MMC). The MMC
generally enters meiosis I, but fails to complete the reductional
phase (apomeiosis), and then undergoes normal meiosis
II to form a nonreduced restitution nucleus (Taraxacum-type
diplospory) (Böcher, 1951; Naumova et al., 2001; Taskin
et al., 2004). In other forms of apomixis, the embryo either
develops from the nucellus (apospory) or else forgoes meiosis
(Antennaria-type diplospory) (Crane, 2001). Third, Boechera
apomicts are pseudogamous, meaning that fertilization of the
central cell is still required for normal endosperm development
(Böcher, 1951; Naumova et al., 2001; Taskin et al., 2004).
Pseudogamy is usually only found in conjunction with apospory
(Richards, 1986; Asker & Jerling, 1992), and typically occurs
in lineages that descend from outbreeding taxa (Mogie, 1992).
Finally, apomixis in Boechera is incomplete (facultative apomixis),
with sexual reproduction still occurring at an often high frequency
(Böcher, 1951; Schranz et al., 2005). Both the formation of
viable pollen and facultative apomixis allow for potential
hybridization with sexual Boechera lineages.
The ability to cross sexual and asexual lineages of Boechera is
critical for the establishment of a genetically tractable research
New Phytologist (2006) 171: 425–438 www.newphytologist.org © The Authors (2006). Journal compilation © New Phytologist (2006)
system. Many of the major advances in our understanding of
the genetic control of apomixis have come from analysis of the
progeny from such crosses (Ozias-Akins et al., 1998; van Dijk
et al., 1999; Noyes & Rieseberg, 2000). However, there are
several important caveats for analysis of the inheritance of
apomixis from such studies, including the problems of making
interploidy and/or interspecific crosses (discussed by Bicknell
& Koltunow, 2004; Matzk et al., 2005). In Boechera we can
partially mitigate these complications. The presence of diploid
apomicts means that we can avoid making interploidy crosses
(Schranz et al., 2005). Also, a robust phylogenetic hypothesis
exists for the relationship of sexual and apomictic lineages,
with direct evidence of past hybridization events (Sharbel &
Mitchell-Olds, 2001; Koch et al., 2003; DobeS et al., 2004a,
2004b; Schranz et al., 2005). Specifically, the sexual species
Boechera stricta is almost exclusively diploid, sexual, and forms
a well supported monophyletic clade (lineage II of DobeS
et al., 2004b; Schranz et al., 2005). Boechera holboellii is
diploid or triploid, often apomictic (Sharbel et al., 2004,
2005), paraphyletic (I. A. Al-Shehbaz, pers. comm.), and is
scattered between two other lineages with other species of Boechera
(lineages I and III of DobeS et al., 2004b; Schranz et al., 2005).
The interspecific hybrid species Boechera divaricarpa has repeated
independent origins by hybridization of B. stricta and B. holboellii-
like plants (Koch et al., 2003; DobeS et al., 2004a, 2004b). These
B. divaricarpa lineages are diploid or triploid, and are often
apomictic (Schranz et al., 2005). Thus apomictic lineages of
B. divaricarpa are known to have a mixed dosage of B. stricta
and B. holboellii-like chromosomes. By making crosses between
diploid B. stricta and B. divaricarpa, we are mirroring the evo-
lutionary steps that have probably happened within the group.
We reported previously on reciprocal crosses between a
sexual B. stricta line (SAD12 = ES6) and a number of diploid
apomictic B. divaricarpa lines, including individuals from the
Vipond Park site (VP9 = ES9) (Schranz et al., 2005). Analysis
of the reciprocal crosses of B. stricta SAD12 and B. divaricarpa
VP9 found that all F1 progeny were diploid and apomictically
derived when VP9 was the maternal parent, whereas all F1
progeny were triploid and sexually derived when SAD12 was
the maternal parent (Schranz et al., 2005).
In this work, we continue our genetic analysis of these
genotypes. First, we provide additional information about the
diploid apomictic parent (VP9), including analysis of male
meiosis, pollen analysis showing nonreduced gamete production,
cytology documenting the presence of a heterochromatic (Het)
chromosome, microsatellite analysis of progeny derived from
self-pollination demonstrating persistent heterozygosity, and
genomic in situ hybridization (GISH) analysis showing its
hybrid origin. From the cross we analysed the F1, F2 progeny,
selected F3 and test-cross (TC) progeny using a combination
of flow cytometry, karyotype analysis, genome painting (GISH),
and seed and pollen grain analyses. In particular, we were interested
in testing the hypothesis that the Het chromosome would act
as a dominant locus and confer the apomictic phenotype

on transmission. Considering Boechera’s great potential for
studies of apomixis, this study serves as a first genetic
investigation into the control and transmission of apomixis in
the group, and has important consequences for our understand-
ing of the evolutionary history of apomixis and hybridization.
Materials and Methods
Plant materials
Analyses of reciprocal crosses made between a common sexual
diploid B. stricta (Graham) Al-Shehbaz tester (SAD12) and a
number of Boechera lines, including apomictic diploid B.
divaricarpa (A. Nelson) A. Löve & D. Löve lines from the
Vipond Park (VP9) site, have been described (Schranz et al.,
2005). In this study we have characterized the SAD12 × VP9
cross in greater detail (Table 1; Fig. 1). We analysed the
parental genotypes [SAD12 (ES 6) and VP9 (ES 9)]; two of
the resulting F1 progeny (ES 116 and ES 117); 20 of their F2
progeny (including lines ES 136 and ES 137); 48 and 30 F3
progeny from lines ES 136 and ES 137, respectively; and test
crosses involving ES 136 and ES 137 (Table 1; Fig. 1).
For the test crosses, the ES 136 and ES 137 lines were used
as maternal parents, and a naturally occurring tetraploid B. stricta
(ES 138) as the paternal pollen-donating parent. The ES 138
line of B. stricta was collected from the Quebec–Labrador
border in Canada, on the north shore of the Strait of Belle
Isle, by John E. Maunder and Nathalie Djan-Chékar of the
Provincial Museum of Newfoundland (collection number NDC
99–348, Stn NDC Bor. 3; accession number NFM 3363).
Plant growth conditions
Seeds were germinated as described by Schranz et al. (2005) and
transplanted into 11 × 11 × 13-cm pots. Plants were grown in
Fig. 1 Pedigree and development of the Boechera SAD 12 × VP9
population.
© The Authors (2006). Journal compilation © New Phytologist (2006) www.newphytologist.org
Research 427
a controlled growth room under long-day conditions (16 h light,
8 h dark). Days to flowering were measured as the number of days
between seedling planting until the appearance of the first open
flower. The ES138 line required a 6-wk vernalization treatment
at 4°C to induce flowering. While we were successful in using ES
138 for genetic crossing, the plant perished before we collected
measurements on the production of pollen or selfed seed.
Chromosome preparation
Fast-growing root tips were collected between 09:00 and 10:00 h
and incubated in a 2-mM aqueous solution of 8-hydroxyquinoline
at 15°C for 3 h, then fixed in ethanol/acetic acid (3 : 1) at
4°C. Meiotic preparations were made of spread meiotic cells
from inflorescences with anthers containing pollen mother
cells at meiosis. The antheres were collected and fixed in acetic
acid : ethanol (1 : 3). To make the microscope slides, we rinsed
the material three times in water and once in 10 mM citrate
buffer pH 4.5, then transferred the material to a pectolytic
enzyme mixture (0.3% pectolyase, 0.3% cellulase RS,
0.3% cytohelicase in 10 mM citrate buffer) for 2 h (meiotic
material, 2.5 h) at 37°C. The root-tip or anther tissues were
washed again, and with fine needles we dissected the root tip
meristem in a small drop of 60% acetic acid on the slide, while
heating carefully on a hot plate at 45°C for 60–90 s. The
material was spread on the microscopic slide by dropping approx.
2 ml freshly prepared ethanol : acetic acid (3 : 1) onto the cells
in the acetic acid solution, followed by a brief dehydration in
ethanol 98% and air-drying. We selected the best chromosome
spread preparations under the phase-contrast microscope.
All chromosome preparations were counterstained with
20 µl 0.2 µg ml−1 4′,6-diamidine-2-phenylindole (DAPI)
in Vectashield Mounting Medium (Vector Laboratories,
Burlingame, CA, USA). Selected mitotic cell complements were
captured for quantification and karyotype analysis. Lengths of
chromosome arms, heterochromatic regions and secondary
constrictions (nucleolus organiser regions, NORs) were established
with the measuring tool of Adobe PHOTOSHOP, and statistical
analyses were performed in Microsoft EXCEL. For karyotype
analysis, we cut out individual chromosomes digitally, ordering
the short arm upwards and matching chromosomes where
possible on the basis of length, centromere position, hetero-
chromatin pattern and presence of satellites/NORs.
Genome painting
We isolated total genomic DNA from the diploid sexuals
B. holboellii (Hornemann) A. Löve & D. Löve, BH208; B. stricta,
BS2; and A. thaliana (accession Columbia) with the Nucleon
Phytopure extraction kit (Amersham Biosciences, Amersham,
UK). DNA was labelled with either digoxigenin-11-dUTP or
biotin-16-dUTP using the nick-translation kit of Roche (Roche
Diagnostics GmbH, Roche Applied Science, Mannheim,
Germany). In the prehybridization step, we first dried the
New Phytologist (2006) 171: 425–438
428 Research

Table 1 Information on plants from the Boechera SAD12 × VP9 population, including generation, line, chromosome number, genome size, days
to flowering, seed set and pollen viability

Chromosome Genome Days to Seeds Pollen

Generation Line number sizea floweringb per fruitc viability (%)d

Parental ES6 14 0.47 288 108.48 98.99

Parental ES9 14 0.50 122 77.56 72.82
F1 ES117 21 0.74 116 0.14 24.52
F1 ES116 21 0.75 101 nd
F2 ES116 #1 nd 0.67 75 nd
F2 ES116 #2 24 0.82 199 0.02
F2 ES117 #1 22 0.80 83 0.02
F2 ES117 #2 23 0.80 291 0.00 40.00
F2 ES117 #3 = ES136 27 0.96 102 10.66 75.88
F2 ES117 #4 24 0.86 129 0.16
F2 ES117 #5 nd 0.72 113 0.62 16.79
F2 ES117 #6 21 0.75 135 0.04 3.43
F2 ES117 #7 28 1.02 139 1.86
F2 ES117 #8 23 0.79 nd nd
F2 ES117 #9 mosaic, 24, 27 1.12 94 0.02
F2 ES117 #10 = ES137 27 0.98 91 9.53 72.81
F2 ES117 #11 nd 0.79 182 0.14
F2 ES117 #12 nd 0.88 206 nd
F2 ES117 #13 nd 0.67 99 0.00
F2 ES117 #14 22 0.76 105 0.04
F2 ES117 #15 21 0.74 101 0.00 1.87
F2 ES117 #16 21 0.73 200 2.66 33.21
F2 ES117 #17 27 0.99 81 0.06
F2 ES117 #19 nd 0.91 92 0.02
F3 ES 136 #3 nd 1.00 150 7.82
F3 ES 136 #9 nd 0.97 113 17.00 70.79
F3 ES 136 #10 nd 0.97 144 8.02 76.41
F3 ES 136 #16 nd 1.01 81 19.62 67.23
F3 ES 136 #24 nd 0.99 84 12.14 64.28
F3 ES 136 #30 nd 0.99 140 46.88 75.58
F3 ES 136 #34 nd 0.99 240 14.84 80.51
F3 ES 137 #9 nd 1.03 145 14.42 71.61
F3 ES 137 #10 nd 0.98 98 5.65 54.66
F3 ES 137 #14 nd 1.04 73 10.13 66.18
F3 ES 137 #15 nd 0.99 112 18.30 68.76
F3 ES 137 #16 nd 0.99 96 6.55 73.09
Tester ES 138 nd 1.01 No floweringe nd
Test cross ES 136 × ES 138 nd 0.95 No floweringf nd
Test cross ES 136 × ES 138 nd 0.97 No floweringf nd
Test cross ES 136 × ES 138 nd 0.99 No floweringf nd
Test cross ES 136 × ES 138 nd 1.01 No floweringf nd
Test cross ES 136 × ES 138 nd 1.03 No floweringf nd
Test cross ES 136 × ES 138 nd 0.99 No floweringf nd
Test cross ES 136 × ES 138 nd 0.92 No floweringf nd
Test cross ES 136 × ES 138 nd 1.04 No floweringf nd
Test cross ES 137 × ES 138 nd 0.96 No floweringf nd
Test cross ES 137 × ES 138 nd 0.97 No floweringf nd

a
Genome size presented as ratio compared with Brassica rapa standard.
b
Days to flowering of nonvernalized plants measured from transfer of seedlings to soil until appearance of first open flower.
c
Seed set per silique calculated by averaging number of seeds from five independent replicates of 10 siliques each.
d
Pollen viability was assessed for selected lines by staining with Alexander’s stain.
e
The tester line did not flower unless vernalized, so days to flowering, seed set and pollen viability were not assessed.
f
The test-cross lines did not flower following 300 d after transplant, so days to flowering, seed set and pollen viability were not assessed.

New Phytologist (2006) 171: 425–438 www.newphytologist.org © The Authors (2006). Journal compilation © New Phytologist (2006)

microscopic preparations at 67°C for 30 min before incubation
with 1 µg ml−1 RNAse-A in 2 × SSC at 37°C for 1 h, and two
wash steps in 2 × SSC for 5 min at 20°C. The preparations
were rinsed in 10 mM HCl for 2 min and then treated with
100 µl pepsin (5 µg ml−1) in 10 mM HCl for 5 min at 37°C,
washed three times in 2 × SSC for 5 min, and fixed in 10%
formaldehyde for 10 min followed by two washes in 2 × SSC
for 5 min (all steps at 20°C). The preparations were dehydrated
in ethanol series (70%, 90% and absolute ethanol for 3 min each)
and air-dried. For single-colour genome painting, we used a
hybridization mixture of 50% formamide, 10% sodium dextrane
sulphate, 2 × SSC, 0.25% SDS and 100 ng DNA probe (BH208
or BS2 genomic DNA) and 10 µg blocking DNA, in a total
of 40 µl hybridization buffer. For the two-colour genome
painting (GISH), the hybridization mixtures contained both
BH208 and BS2 probes and blocking DNA (1 : 100 total
genomic DNA of A. thaliana). The mixture was denatured at
100°C for 10 min and chilled immediately on ice for 10 min
before use. For each preparation we added 40 µl of the
hybridization mix and covered it with a 24 × 50 mm cover
slide, followed by heating on an 80°C hot plate for 2.5 min before
an overnight hybridization at 37°C in a humidified chamber.
Post-hybridization washes involved three wash steps of 50%
formamide in 2 × SSC (pH 7.0) at 42°C for 5 min, and two
steps in 2 × SSC for 5 min at 20°C. The hybridization signals
were detected with fluorescein isothiocyanate (FITC)-conjugated
anti-DIG antibodies and amplified with FITC-conjugated
rabbit anti-sheep antibodies for the digoxigenin-labelled probe,
and with Avidin Texas-Red for the biotin-labelled probe that
was amplified with biotinylated antiavidine and Avidin Texas-
Red. The preparations were counterstained in 100 µl of a 2 µg
ml−1 DAPI solution in 100 mM citrate buffer pH 6.0 for 10
min in the dark, and finally mounted in Vectashield (Vector
Laboratories) under a 24 × 50-mm cover slip. Chromosomes
were examined under a Zeiss Axioplan 2 photomicroscope
equipped with epifluorescence illumination and filter sets
for DAPI, FITC and Texas Red fluorescence. The images were
captured with a Photometrics Sensys 1305 × 1024 image array
CCD camera and analysed using GENUS ver. 2.7 IMAGE ANALYSIS
WORKSTATION software (Applied Imaging International Ltd.,
Newcastle upon Tyne, UK). DAPI images were sharpened with
a 7 × 7 Hi-Gauss high-pass spatial filter to accentuate minor
details and heterochromatin differentiation of the chromosomes.
We used the levels and curves tools in Adobe PHOTOSHOP to
improve DAPI heterochromatin differentiation banding, and
used the saturation tool to enhance colour saturation of the
green and red fluorescence signals.
DNA extraction, microsatellite amplification and
analysis
The isolation of DNA, microsatellite amplification and analysis
were carried out as described by Schranz et al. (2005). Ampli-
fications were done with microsatellites (GC27, ICE3, Bf-3,
© The Authors (2006). Journal compilation © New Phytologist (2006) www.newphytologist.org
Research 429
ICE14, H23) that were identified as being heterozygous
with two alleles each (bi-allelic) in the VP9 genotype (Schranz
et al., 2005). Based on sequence similarity to Arabidopsis and
preliminary genetic mapping results (M.E.S. and T.M.O.,
unpublished data) these loci are unlinked. Ten self-derived
progeny from VP9, eight test-cross lines from ES136, and two
test-cross lines from ES137 were analysed.
Genome-size measurements
Ploidy analyses were performed on a PARTEC (Münster,
Germany) CCA-II flow cytometer using their CyStain UV
precise P nuclei extract and staining kit (PARTEC GmbH)
according to the manufacturer’s protocol. Sample leaf material
was measured in combination with an internal size standard
(leaf material from Brassica rapa and/or Matthiola incana for
larger genome individuals), and the ratio of the mean fluores-
cence intensity values for the 2c peaks for both sample and
standards were calculated (Schranz et al., 2005). When Matthiola
was used as standard, the ratio was converted to the Brassica units
(by using the size ratio of Matthiolia compared with Brassica).
A minimum of 10 000 fluorescence counts was collected for
each sample run.
Seed collection and measurement
Seed collections were made from mature plants. Ten siliques
from each of five mature reproductive axes per plant were
collected (for a total of 50 siliques). The number of seeds from
each group of 10 siliques was counted and the average number
of seeds per fruit calculated (Table 1; see Fig. 6).
Pollen grain staining and measurement
Measurements of pollen counts and viability were estimated
using a modified Alexander’s stain (Alexander, 1980). Pollen
grains were counted by collecting two to five freshly opened
flowers from each plant. The Alexander stain that gave the
best results contained the minimum amount of lactic acid
(0.5 ml), suggested for thin-walled pollen (Alexander, 1980).
A dilution of 1 : 20 Alexander stain stock solution to 50%
glycerin was used. A total of 20 µl stain dilution was placed
on an object slide. An individual stamen from each flower
was put on a microscopic slide containing a drop of the stain
dilution, and covered with a cover slip. We left the material
for at least 10 min before analysing the pollen grains. The
numbers of viable (purple) and nonviable (blue/green) pollen
grains were recorded using an Axioskop 2 compound microscope
(Carl Zeiss GmbH, Jena, Germany) at ×20 magnification.
Between 400 and 2000 pollen grains for each plant were counted,
from which the percentage of viable pollen was calculated (Table 1).
The sizes of both viable and nonviable grains were also
measured using the stained pollen grains. Photos of the pollen
grains were made with a Zeiss AxioCam camera and using the
New Phytologist (2006) 171: 425–438
430 Research
program AXIOVISION ver. 3.0.6.1 (Carl Zeiss Vision GmbH,
Hallbergmoos, Germany). The area of pollen grains was
estimated from the photos using the IMAGEJ ver. 1.33µ program
(Abramoff et al., 2004) from which the diameter of each
grain was calculated. Between 20 and 44 individual photos,
representing between 94 and 825 individual pollen grains per
plant, were analysed (see Fig. 7).
Results
Genome composition revealed by GISH
Genomic in situ hybridizations (GISH) were initially done with
total genomic DNA from the diploid sexual B. stricta labelled
as a probe (red) and blocked with genomic DNA from B. holboellii
(Fig. 2a–d). Chromosome complements showed fluorescent
signals on the pericentromere regions of only the seven B. stricta-
derived chromosomes in the hybrid B. divaricarpa VP9 genome
(Fig. 2c), confirming its homoploid origin (hybridization with
no increase in ploidy) between a B. stricta- and B. holboellii-like
plant. Additionally, the hybrid B. divaricarpa VP9 genome was
found to contain a highly heterochromatic chromosome (Het)
(indicated by arrows in Fig. 2a,c) that may have properties
similar to B-chromosomes and/or Het chromosomes identified
in 15-chromosome Boechera apomicts (Sharbel et al., 2004;
Kantama, 2005). The fluorescent signal on the Het chromosome
(Fig. 2c) suggests it is derived from the B. stricta ancestor of
this hybrid line. Genome painting of ES 117 F1 line chromo-
somes (Fig. 2d) further revealed a total of 14 B. stricta chromo-
somes (seven derived from a reduced haploid B. stricta SAD12
gamete; the other seven from the nonreduced B. divaricarpa
VP9 gamete) and seven B. holboellii-like chromosomes (seven
derived from the nonreduced B. divaricarpa VP9 gamete).
An additional two-colour genome painting was carried
out with the simultaneous hybridization of both B. stricta
(green) and B. holboellii (red) probes, and blocking with total
genomic DNA of A. thaliana in order to further improve the
discrimination of the parental species (Kantama, 2005). The
painting of one of the highly fertile F2 lines, ES 137, revealed
14 B. stricta chromosomes (green), including the highly
heterochromatic (Het) chromosome (arrow in Fig. 2e), and
13 B. holboellii-like chromosomes (Fig. 2e). Thus the two
likely aneuploid gametes derived from the ES 117 line (with
its 14 B. stricta and seven B. holboellii-like chromosomes) that
united to form the ES 137 plant were effectively able to restore
the chromosome balance of one B. stricta to one B. holboellii-like
chromosome (Fig. 2e). The restored genomic ratio could explain
the higher fertility of this line. However, we do not yet know
if we have complete chromosome complements in these lines.
Analysis of pollen meiosis of VP9
Anther preparations of VP9 showed very few meiocytes. We
found pachytene and metaphase I complements with fully
New Phytologist (2006) 171: 425–438 www.newphytologist.org © The Authors (2006). Journal compilation © New Phytologist (2006)
paired bivalents. Metaphase I cells diplayed seven regular
bivalents, showing that meiosis in this plant can be fully
synaptic. At this stage we could identify the Het chromosome
in a few cells forming a heteromorphic bivalent with one of
the other chromosomes (arrow, Fig. 3). We did not find any
anaphase I or anaphase II complement.
Microsatellite analysis of self-progeny of VP9 and
test-cross lines
In our previous study (Schranz et al., 2005) we analysed micro-
satellites that were bi-allelic in VP9. In a sample of 10 progeny
derived by self-pollination of VP9, heterozygosity was maintained
(Fig. 4). The probability of obtaining all heterozygous genotypes
by sexual reproduction with independent assortment is very
unlikely (P < 0.001). This result strongly supports the conclusion
that VP9 is an apomictic lineage. The microsatellites used were
also highly polymorphic between VP9 and both SAD12 and
ES138. The test-cross lines, where ES136 and ES137 were
used as maternal plants and ES138 as a pollen donor, all
contained alleles derived from ES138. This result, and the
dramatic change in phenotype (exemplified by the shift to very
late flowering time), both indicate that the near-tetraploid
lines ES136 and ES137 reproduce sexually.
Genome size, chromosome numbers and karyotypes
Flow cytometry results of all lines were standardized to the
diploid B. rapa genome (Table 1), and chromosome counts
were done for most of the parental, F1 and F2 plants (Table 1).
Comparison of flow cytometry values with chromosome counts
(assuming all chromosomes are of approximately the same
size) showed very good correspondence (R 2 = 0.98) (Fig. 5a).
Chromosome counts and karyotype analysis (Fig. 5b) confirmed
that the parental genotypes were diploid (2n = 2x = 14).
The F1 plants (ES 116 and ES 117) were also confirmed to be
triploids (2n = 3x = 21) (Fig. 5b), as hypothesized from earlier
flow cytometry results, probably caused by the union of a reduced
and nonreduced gamete (Schranz et al., 2005)
The most striking result was the swarm of chromosome
numbers of the F2 lines, ranging from triploid to tetraploid,
with most being aneuploid (Table 1; Fig. 5a). While overall
genome-size measurements agreed with the chromosome
counts, there was one notable exception. The F2 line ES 117
#9 had the largest genome size measured by flow cytometry
(1.12), but gave a mosaic of aneuploid chromosome numbers
(24 and 27). Two F2 lines, ES 136 and ES 137, were found to
be highly fertile (see below). Chromosome counts for both
these lines showed that they were nearly tetraploid (2n ≈ 4x ≈
27) (Table 1; Fig. 5b). Because of their fertility, we investi-
gated the genome size of their F3 progeny. All F3 progeny had
genome sizes of the same magnitude (ranging from 0.94 to
1.08) (Table 1; Fig. 5c), suggesting that the genomes of these
two lines have stabilized near tetraploidy.
 Research 431

Fig. 2 Genome composition revealed by genomic in situ hybridization

(GISH) of selected Boechera SAD12 × VP9 population lines. DAPI
staining of chromosomes of: (a) paternal apomictic Boechera
divaricarpa genotype (VP9); (b) F1 line (ES 117). Fluorescence in
situ hybridizations done with total genomic DNA from the diploid
sexual Boechera stricta labelled as a probe (red) and blocked with
genomic DNA from Boechera holboellii staining the pericentromere
regions of: (c) seven B. stricta-derived chromosomes in the hybrid
B. divaricarpa (VP9) genome; (d) 14 B. stricta-like chromosomes in
the F1 line (ES 117) genome (arrows in Fig. 5(a,c) denote the Het
chromosome). Additional two-colour genome painting done by
the simultaneous hybridization of both B. stricta (green) and B.
holboellii (red) probes onto (e) one of the high-fertility F2 lines, ES
137, showing 14 B. stricta chromosomes (green) including a highly
heterochromatic chromosome (arrow) and 13 B. holboellii-like
chromosomes.

© The Authors (2006). Journal compilation © New Phytologist (2006) www.newphytologist.org New Phytologist (2006) 171: 425–438
432 Research
Fig. 3 DAPI-stained chromosome spread of anther cells of VP9.
Metaphase I complement with seven bivalents showing that meiosis
in this plant is fully synaptic. Arrow, Het chromosome in the
heteromorphic bivalent.
Fig. 4 Microsatellite amplification of 10 progeny derived by self-
pollination of VP9. At several unlinked microsatellite loci, including
this locus (GC27), the initial VP9 plant was heterozygous. In a sample
of 10 progeny derived by self-pollination of VP9, heterozygosity was
maintained for all samples, suggesting that progeny were derived
apomictically rather than sexually.
The strong correlation between the flow cytometry results
and chromosome number allowed us to estimate the genome
size of Boechera. A recent study (Johnston et al., 2005) estimated
the haploid genome of B. rapa (n = 10) at 1C = 529 Mb. Using
this estimate, the haploid Boechera genome (n = 7) would be
approx. 264 Mb. However, their estimate for the size of the
A. thaliana genome is 157 Mb ( Johnston et al., 2005), which
is larger than current estimates based on genome sequencing.
Using the latter value, we infer that the haploid Boechera
genome is roughly 1.7 times that of A. thaliana.
Seed production of aneuploid and polyploid F2 lines
Analysis of seed production of F1 and F2 plants found that most
were highly infertile (Table 1; Fig. 6). However, two F2 lines,
ES 136 and ES 137, had much greater seed set with c. 10 seeds
per silique, compared with an average of only 0.36 seeds per
silique for the other lines (Table 1; Fig. 6). Correlation between
genome size and seed set (Fig. 6) was not significant, as
New Phytologist (2006) 171: 425–438 www.newphytologist.org © The Authors (2006). Journal compilation © New Phytologist (2006)
most lines of varying genome sizes were infertile, but the two
higher-fertility lines were nearly tetraploid. The F3 lines
derived from ES 136 and ES 137 all maintained high levels of
fertility, with an average of 15.11 seeds per fruit; however,
there was still great variation in seed production (5.65–48.88
seeds per silique; Table 1).
Pollen grain viability and size
The viability of pollen grains differed between the two parental
lines, with sexual B. stricta SAD12 producing almost all viable
pollen, and the apomictic B. divaricarpa VP9 producing 74.88%
viable pollen (Table 1). The triploid F1 plant (ES 117) had greatly
reduced pollen viability of 24.52% (Table 1). The swarm of
F2 plants displayed great variation in viability, from only
1.87% up to 75.88 and 72.81% for lines ES 136 and ES 137,
respectively (Table 1). The F3 progeny of ES 136 and ES 137
maintained the higher-viability pollen, with an average of
69.92% (Table 1).
The analysis of pollen grain size of viable and nonviable pollen
revealed several striking results (Fig. 7). The sexual B. stricta
SAD12 produced almost exclusively reduced and viable
pollen grains with an average diameter of 16.53 µm (Fig. 7a).
The apomictic B. divaricarpa VP9 produced viable, nonreduced
pollen grains with an average diameter of 22.32 µm. Interest-
ingly, VP9 also produced some pollen grains of even larger
diameter (Fig. 7a). The F1 line ES 117 produced viable pollen
grains of varying size, from 17.66 to 25.82 µm (Fig. 7a), probably
reflecting a wide range of aneuploid gametes. However, no
viable gametes of haploid size were detected. The nonviable
pollen of ES 117 (Fig. 7b) was found to be smaller than that
of the viable pollen. The size range of the nonviable pollen was
from 12.51 to 22.31 µm, corresponding to gametes that would
be less than haploid to about diploid in size. The F2 line, ES
117 #15, was triploid and produced almost exclusively non-
viable pollen (1.87% viability; Table 1). The size distribution
of the nonviable grains for ES 117 #15 (Fig. 7b) ranged from
11.92 to 19.90 µm, and thus were smaller than diploid in size.
Finally, one of the F3 lines derived from ES 137 (ES 137 #15)
produced viable pollen grains from 18.89 to 25.09 µm, and
nonviable pollen grains from 13.37 to 20.87 µm in diameter
(Fig. 7a,b).
The six lines discussed above (Fig. 7a,b) illustrate the
variability in pollen viability and size from the various genera-
tions. In addition, overall pollen grain viability and size for all
nonparental lines was also determined (Fig. 7c). The average
size of nonviable gametes was 15.88 µm, which is slightly less
than that of haploid, and far less than the 21.91 µm of the
diploid viable gametes (Fig. 7c). It should be noted that the
ratio of nonviable to viable pollen grains measured for size was
lower than that obtained by simply counting nonviable and
viable pollen grains. This discrepancy was because nonviable
pollen grains clumped together and thus it was difficult to
take good photographs for size measurements.
 Research 433

Fig. 5 Measures of genome size of lines in

the Boechera SAD12 × VP9 population.
(a) Correlation of genome size (measured by
flow cytometry and given as ratio to reference
Brassica rapa genome) and chromosome
numbers. (b) Karyotypes and chromosome
numbers of selected generations and lines.
(c) Distribution in genome size (measured by
flow cytometry and given as ratio to reference
B. rapa genome) of the F3 lines derived from
ES136 and ES137, demonstrating the genomic
stability of these lines near tetraploidy.

Discussion
Here we present the first genetic analysis of the mechanisms
and inheritance of apomictic reproduction in Boechera. First,
we characterized more fully the paternal diploid apomictic
line VP9. Genome painting revealed its hybrid origin, whereas
the analysis of male meiosis demonstrated that the chromosomes
are fully synaptic, including a heterochromatic (Het) chromosome
that fully pairs with one of the other chromosomes. Male meiosis
produced both reduced and unreduced gametes, suggesting
first- or second-division restitution. Heterozygosity was also
maintained in self-pollinated progeny, thus supporting its
apomictic nature. We wondered if this Het chromosome carries
the genetic elements controlling apomixis in a manner similar
to several other systems where supernumerary or hemizygous
chromosomes carry an apomixis factor (Ozias-Akins et al.,

© The Authors (2006). Journal compilation © New Phytologist (2006) www.newphytologist.org New Phytologist (2006) 171: 425–438
434 Research

Fig. 6 Comparison of genome size (measured

by flow cytometry and given as ratio to
reference Brassica rapa genome) and seed
production of F2 lines of the Boechera
SAD12 × VP9 population. Most lines are
highly infertile except two tetraploids (ES 136
and ES 137) with greater seed production.

1998; Bicknell et al., 2000; Roche et al., 2001; Labombarda
et al., 2002; Akiyama et al., 2005). Therefore we analysed the
progeny derived from a cross between a diploid sexual and this
diploid apomictic line. The resulting triploid F1 was largely
sterile, producing limited numbers of viable pollen and seed.
When the few seeds were grown, they produced plants that
were mostly aneuploid or near-tetraploid. Hence they were
not apomictically derived replicates of the three maternal
genomes, but rather the product of a reduced or unreduced
meiosis. Two of the recovered lines were fertile, sexual and
near-tetraploid. Genome painting (GISH) analysis of one
of these fertile lines found that it had a nearly balanced
chromosome complement of 14 B. stricta and 13 B. holboellii
chromosomes, suggesting that genome balance may play an
important role in establishing the sexual phenotype. However,
future work will be needed to establish if these correspond
to complete chromosomal complements. Below we discuss
in greater detail some of the implications of our results for
understanding the inheritance, control and evolution of
apomixis in Boechera, and the potential importance of the
generation of sexual tetraploid lineages.
Control of apomixis in Boechera
Asexual seed formation is a complex trait resulting from modi-
fication of the sexual life cycle (reviewed by Koltunow &
Grossniklaus, 2003; Bicknell & Koltunow, 2004). The normal
sexual pathway can be altered or deregulated by the inheritance
of a single dominant gene, hemizygous genomic region, and/
or supernumerary element (Nogler, 1984b; Sherwood et al.,
1994; Bicknell et al., 2000; Roche et al., 2001). Our F1 plants
were derived from the union of a reduced egg cell from the
diploid sexual maternal plant and a nonreduced pollen grain
from the apomictic paternal plant, as shown by GISH analysis.
Hence the F1 progeny probably inherited the complete genome
of the apomictic parent. However, there may have been cross-
over events during the failed meioses of the paternal VP9 plant
that could have produced recombinant pollen grains. Future
segregation analyses of molecular markers will be necessary to
examine this possibility.
Our chromosome analyses showed that the diploid apomictic
paternal plant had an aberrant chromosome resembling
the heterochromatic (Het) chromosome in other 14- and 15-
chromosome Boechera apomicts (Kantama, 2005), but lacked
the extra chromosome found in the 15-chromosome apomicts
(Sharbel et al., 2004, 2005). The offspring individuals in
the F2 progeny displayed varying aneuploid and polyploid
chromosome numbers, suggesting that apomixis in Boechera
is not regulated by the simple inheritance of a single dominant
locus or factor (e.g. the Het chromosome). If it were, then
the F1 line, which contains the Het chromosome, would be
apomictic with all or most derived F2 progeny being identical
to the maternal plant.
Various authors have pointed to polyploidy as being indis-
pensible in the regulation and transmission of the apomixis trait
(Quarin et al., 2001). But in Boechera, where naturally occurring
triploids are indeed apomictic, they can be highly facultative
with many progeny derived sexually (Schranz et al., 2005). The

Fig. 7 Histograms of Boechera pollen grain size and viability. Alexander’s stain was used to differentiate viable (purple) and nonviable (blue/
green) pollen grains, and the diameter of each grain was measured by analysis of photos of pollen. Frequency based on total number of pollen
grains counted. Size distribution and selected pollen grain images of (a) viable; (b) nonviable pollen grains of six selected lines used to illustrate
the diversity of viability and size of the various lines. The lines used were SAD12 (diploid sexual); VP9 (diploid apomict); ES117 (triploid F1);
ES137 (near-tetraploid F2); ES117 #15 (aneuploid F2); ES137 #15 (near-tetraploid F3). (c) Size distribution of all viable and nonviable pollen grains
measured from aneuploid and polyploid lines (excluding the two diploid parental lines).

New Phytologist (2006) 171: 425–438 www.newphytologist.org © The Authors (2006). Journal compilation © New Phytologist (2006)
 Research 435

© The Authors (2006). Journal compilation © New Phytologist (2006) www.newphytologist.org New Phytologist (2006) 171: 425–438
436 Research
results of our crossing have also shown that an increase in
ploidy from diploidy to triploidy is not necessarily associated
with the transfer or expression of the apomictic phenotype.
It has also been postulated that the hybrid constitution of
apomictic lineages, rather than polyploidy, will lead to expres-
sion of the apomictic phenotype (Bicknell & Koltunow, 2004).
Specifically, the hybridization-derived floral asynchrony hypo-
thesis postulates that apomixis is caused by the differential
expression and temporal regulation of genes in normal
sexual reproductive pathways (Carman, 1997; Koltunow &
Grossniklaus, 2003). In agreement with this hypothesis is our
demonstration by GISH that the diploid apomictic parent
(VP9) is of hybrid origin, with seven B. stricta and seven B.
holboellii-like chromosomes. In addition, all diploid and triploid
lines that were apomictic and/or produced nonreduced
gametes were also highly heterozygous, attesting to their likely
hybrid origin (Schranz et al., 2005). Similarly, individuals
found to be heterozygous at microsatellite loci (DobeS et al.,
2004b) and at a sequenced molecular marker (Sharbel et al.,
2004, 2005) tended to produce nonreduced gametes, and
were concluded to be apomictic.
However, hybridization alone cannot explain apomixis in
Boechera. Our triploid F1, which contained a complete haploid
genome from the highly inbred maternal B. stricta plant and
the potentially complete diploid B. divaricarpa genome from
the apomictic paternal plant, is not apomictic. One possible
explanation for this failure in transmission of the apomixis
phenotype could be segregation of the apomixis gene(s) caused
by recombination during the production of the 2n pollen
grains in the apomictic paternal plant. Chromosome analysis,
however, showed that all or part of the heterochromatic
(Het) chromosome was transmitted to the F1 plant. Another
explanation for the discrepancy may lie in the relative dosage
of B. stricta and B. holboellii-like chromosomes. GISH analyses
of the diploid apomictic showed there is a 1 : 1 genome ratio,
but in the F1 triploid there is a ratio of 2 : 1 in favour of the
sexual B. stricta genome. The higher dose of B. stricta might allow
for the correct, and disruptive, expression of sexuality in the
triploid. Naturally occurring triploid apomictic lineages may
have two doses of B. holboellii-like chromosomes, or a dosage
closer to 1 : 1 if the triploid apomictic line is derived from the
union of two aneuploid gametes. However, one of the recovered
F2 near-tetraploid lines has a nearly 1 : 1 genome ratio, yet is
sexual. Sexuality at the tetraploid level may be feasible because
of an interaction of dosage and polyploidization, or alternatively
because of the segregation of a gamete-lethality factor.
The results of our pollen analysis may shed light on the
possible segregation of a gamete-lethal factor or gene. In all
samples, except the diploid sexual line, there a significant
fraction of nonviable pollen grains were produced, which
were of approximately the same size as viable haploid pollen.
In contrast, viable pollen grains in all samples, except for the
sexual diploid, were of approximately diploid size. No viable
haploid-sized pollen was observed for any of these lines. Some
New Phytologist (2006) 171: 425–438 www.newphytologist.org © The Authors (2006). Journal compilation © New Phytologist (2006)
of the nonviable pollen grains might be caused simply by
chromosomal imbalances, particularly those generated from
aneuploid F2 lines where the percentages of nonviable pollen
grains were as high as 98%. However, both the apomictic
diploid parent and the recovered sexual tetraploids produced,
on average, 25% nonviable gametes and 75% viable diploid-
sized pollen grains. This result would be consistent with the
recessive lethal gametophytic selection hypothesis of (Nogler,
1984b), in which an apomixis gene or factor is lethal when it
occurs in a haploid pollen grain in the absence of a wild-type
allele. Future studies examining the patterns of inheritance of
molecular and/or cytological markers will be critical for resolving
models for the control of apomixis in Boechera.
Escape from apomixis, low fertility and return to
sexuality
Traditionally, apomictic lineages were thought to have limited
evolutionary potential and to be doomed to extinction (Darlington,
1939; Stebbins, 1950). Later, researchers realized that the
maintenance of male function and facultative apomixis provided
mechanisms for perpetuating apomictic lineages by transferring
apomixis genes via hybridization with sexual relatives (van
Dijk, 2003). The transfer of apomixis genes into new genetic
backgrounds would allow the purging or masking of deleterious
mutations that may have accumulated in the apomict (Muller’s
ratchet), and would generate new genetic diversity, allowing
escape from parasitism. Another possibility is that the hybridiza-
tion of asexuals with sexuals allows genes from the apomictic
parent to re-enter the sexual gene pool (Chapman et al., 2003),
undergo recombination, and potentially go on to form new
apomictic lineages (de Wet, 1968).
We have detected such a shift in breeding system, from
apomict diploid to low-fertility sexual triploid to fertile
and sexual tetraploid, in our experimental cross of Boechera. In
many ways our cross follows the classic model of the formation
of autotetraploids via a triploid bridge, and the inherent problems
of meiosis in triploids and the frequent endosperm develop-
mental problem of the triploid block (Ramsey & Schemske,
1998; Husband, 2004; Henry et al., 2005).
There are two routes from apomictic genotypes to the
formation of sexual tetraploids in Boechera. In this work we
demonstrated that, by crossing the two diploids, we could
generate sexual near-tetraploids in only two generations. In
our earlier work, we frequently derived de novo tetraploids
directly from the crossing of a sexual diploid and an apomictic
triploid line. These sexual tetraploid lines can then undergo
all the classic advantages of sex, such as recombination and
independent assortment. There could also be chances for
homologous pairing, translocations and epigenetic modifica-
tions often seen in new polyploid lines (Osborn et al., 2003),
all generating new phenotypic diversity. Also, it is important
to note that these tetraploid Boechera lines could potentially
express apomixis, or some partial component of apomixis, at

some frequency, caused by the penetration or expressiveness
of the traits. The partial expression of apomixis, especially
parthenogenesis, may be particularly important for the
generation of new diploid apomictic lineages. The establishment
of diploid hybrid apomictic haplotypes in Boechera could be
caused by homoploid hybridization, or alternatively by base
chromosome number reductions (Asker & Jerling, 1992). If
reduced 2n egg cells from tetraploid Boechera lineages develop
parthenogenically, they could establish new diploid lineages
that could be either sexual or apomictic. Such cycles of ploidy
have been described for other apomictic complexes, such as
Potentilla argentea and the Bothriochloa–Dichanthium complex,
and are known as the ‘diploid–tetraploid–dihaploid cycle’ (de
Wet, 1968; Asker & Jerling, 1992). The independent assortment
and recombination of chromosomes derived from the parental
genotypes or species of the tetraploid means that there could
be unusual constellations of B. stricta and B. holboellii chromo-
somes or chromosome regions in dihaploids produced
parthenogenically.
Interestingly, few naturally occurring tetraploid Boechera
lineages have been detected (reviewed by DobeS et al., 2006),
but when they do occur, they are likely to be sexual (Böcher, 1969;
Johnson, 1970; Schranz et al., 2005). The low frequency of
tetraploids could be explained by a potential propensity to
produce dihaploid offspring. Alternatively they may be selected
against, possibly because of a fitness disadvantage. It is inter-
esting to note that sexual tetraploids existing at low frequency
in populations of dandelions have been postulated to be critical
for the creation of new triploid apomictic cytotypes, by the
production of reduced 2n gametes that recombine with reduced
n gametes from sexual diploids (Verduijn et al., 2004). Overall,
the historical occurrence of hybridization shifts in breeding
systems and alterations of ploidy have profound implications
for our understanding of the inheritance, transmission and
evolution of genetic and quantitative variation for Boechera.
Acknowledgements
The authors thank Juliane Pfuetzenreuter, Silke Fuchs and Radim
Vasut for technical assistance. Thanks to John E. Maunder and
Nathalie Djan-Chékar of the Provincial Museum of New-
foundland and Labrador for providing seeds. The authors
also thank Peter van Dijk for helpful comments. Support for
this research was provided by the Max Planck Gesellschaft to
M.E.S. and by the Thai Government to L.K.
References
Abramoff MD, Magelhaes PJ, Ram SJ. 2004. Image processing with
IMAGEJ. Biophotonics International 11: 36 – 42.
Akiyama Y, Hanna W, Ozias-Akins P. 2005. High-resolution physical
mapping reveals that the apospory-specific genomic region (ASGR) in
Cenchrus ciliaris is located on a heterochromatic and hemizygous region
of a single chromosome. Theoretical and Applied Genetics 111: 1042–
1051.
© The Authors (2006). Journal compilation © New Phytologist (2006) www.newphytologist.org
Research 437
Albertini E, Porceddu A, Ferranti F, Reale L, Barcaccia G, Romano B,
Falcinelli M. 2001. Apospory and parthenogenesis may be uncoupled in
Poa pratensis: a cytological investigation. Sexual Plant Reproduction 14:
213–217.
Alexander MP. 1980. A versatile stain for pollen, fungi, yeast and bacteria.
Stain Technology 55: 13–18.
Asker SE, Jerling L. 1992. Apomixis in Plants. Boca Raton, FL, USA: CRC
Press.
Bicknell RA, Koltunow AM. 2004. Understanding apomixis: recent
advances and remaining conundrums. Plant Cell 16: S228–S245.
Bicknell RA, Borst NK, Koltunow AM. 2000. Monogenic inheritance of
apomixis in two Hieracium species with distinct developmental
mechanisms. Heredity 84: 228–237.
Böcher T. 1951. Cytological and embryological studies in the amphil–
apomictic Arabis holboellii complex. Biologiske Skrifter 6: 1–58.
Böcher T. 1954. Experimental taxonomical studies in the Arabis holboellii
complex. Svensk Botanisk Tidskrift 48: 31–44.
Böcher T. 1969. Further studies in Arabis holboellii and allied species.
Saertryk af Tidsskrift 64: 141–161.
Carman J. 1997. Asynchronous expression of duplicated genes in
angiosperms may cause apomixis, bispory, tetraspory, and polyembryony.
Biological Journal of the Linnean Society 61: 51–94.
Chapman H, Houliston GJ, Robson B, Iline I. 2003. A case of reversal:
the evolution and maintenance of sexuals from parthenogenetic clones in
Hieracium pilosella. International Journal of Plant Sciences 164: 719 –728.
Charlesworth B. 1990. Mutation–selection balance and the evolutionary
advantage of sex and recombination. Genetical Research 55: 199–221.
Crane CF. 2001. Classification of apomictic mechanisms. In: Savidan Y,
Carman J, Dresselhaus T, eds. The Flowering of Apomixis: From
Mechanisms to Genetic Engineering. Mexico: CIMMYT, IRD/European
Commission DG VI, 24–43.
Darlington C. 1939. The Evolution of Genetic Systems. Cambridge, UK:
Cambridge University Press.
van Dijk PJ. 2003. Ecological and evolutionary opportunities of apomixis:
insights from Taraxacum and Chondrilla. Philosophical Transactions of the
Royal Society of London, Series B – Biological Sciences 358: 1113–1120.
van Dijk PJ, Tas ICQ, Falque M, Bakx-Schotman T. 1999. Crosses between
sexual and apomictic dandelions (Taraxacum). II. The breakdown of
apomixis. Heredity 83: 715–721.
Dobes CH, Koch MA, Sharbel TF. 2006. Embryology, karyology, and
modes of reproduction in North American genus Boechera (Brassicaceae):
a compilation of seven decades of research. Annals of the Missouri Botanical
Garden. (In press.)
Dobes C, Mitchell-Olds T, Koch MA. 2004a. Intraspecific diversification in
North American Boechera stricta (= Arabis drummondii ), Boechera ×
divaricarpa and Boechera holboellii (Brassicaceae) inferred from nuclear and
chloroplast molecular markers – an integrative approach. American Journal
of Botany 91: 2087–2101.
Dobes CH, Mitchell-Olds T, Koch MA. 2004b. Extensive chloroplast
haplotype variation indicates Pleistocene hybridization, radiation of
North American Arabis drummondii Arabis drummondii, A. × divaricarpa,
and A. holboellii (Brassicaceae). Molecular Ecology 13: 349–370.
Henry I, Dilkes B, Young K, Watson B, Wu H, Comai L. 2005. Aneuploidy
and genetic variation in the Arabidopsis thaliana triploid response. Genetics
170: 1979–1988.
Holsinger KE. 2000. Reproductive systems and evolution in vascular plants.
Proceedings of the National Academy of Sciences, USA 97: 7037–7042.
Husband BC. 2004. The role of triploid hybrids in the evolutionary
dynamics of mixed-ploidy populations. Biological Journal of the Linnean
Society 82: 537–546.
Johnston JS, Pepper AE, Hall AE, Chen ZJ, Hodnett G, Drabek J,
Lopez R, Price HJ. 2005. Evolution of genome size in Brassicaceae.
Annals of Botany 95: 229–235.
Johnson TF. 1970. Investigations in the floral biology of the Arabis holboellii
complex. MSc thesis. Seattle, WA, USA: University of Washington.
New Phytologist (2006) 171: 425–438
438 Research
Kantama L. 2005. Chromosome studies and genetic analyses of natural
and synthetic apomictic model species. PhD thesis. Wageningen,
the Netherlands: Wageningen University.
Koch M, Haubold B, Mitchell-Olds T. 2001. Molecular systematics of
the Brassicaceae: evidence from coding plastidic matK and nuclear
Chs sequences. American Journal of Botany 88: 534 – 544.
Koch MA, Dobes C, Mitchell-Olds T. 2003. Multiple hybrid formation in
natural populations: concerted evolution of the internal transcribed spacer
of nuclear ribosomal DNA (ITS) in north American Arabis divaricarpa
(Brassicaceae). Molecular Biology and Evolution 20: 338 – 350.
Koltunow AM, Grossniklaus U. 2003. Apomixis: a developmental
perspective. Annual Review of Plant Biology 54: 547– 574.
Labombarda P, Busti A, Caceres ME, Pupilli F, Arcioni S. 2002. An
AFLP marker tightly linked to apomixis reveals hemizygosity in a portion
of the apomixis-controlling locus in Paspalum simplex. Genome 45:
513–519.
Leblanc O, Grimanelli D, Gonzalez-d, e-Leon D, Savidan Y. 1995.
Detection of the apomictic mode of reproduction in maize–Tripsacum
hybrids using maize RFLP markers. Theoretical and Applied Genetics 90:
1198–1203.
Matzk F, Prodanovic S, Baumlein H, Schubert I. 2005. The inheritance of
apomixis in Poa pratensis confirms a five locus model with differences in
gene expressivity and penetrance. Plant Cell 17: 13 –24.
Mogie M. 1992. The Evolution of Asexual Reproduction in Plants. New York,
NY, USA: Chapman & Hall.
Naumova TN, van der Laak J, Osadtchiy J, Matzk F, Kravtchenko A,
Bergervoet J, Ramulu KS, Boutilier K. 2001. Reproductive development
in apomictic populations of Arabis holboellii (Brassicaceae). Sexual Plant
Reproduction 14: 195–200.
Nogler GA. 1984a. Gametophytic apomixis. In: Johri B, ed. Embryology
of Angiosperms. Berlin: Springer, 475 – 518.
Nogler GA. 1984b. Genetics of apospory in apomictic Ranunculus
auricomus. 5. Conclusion. Botanica Helvetica 94: 411– 422.
Noyes RD, Rieseberg LH. 2000. Two independent loci control
agamospermy (apomixis) in the triploid flowering plant Erigeron annuus.
Genetics 155: 379–390.
Osborn TC, Pires JC, Birchler JA, Auger DL, Chen ZJ, Lee HS, Comai L,
Madlung A, Doerge RW, Colot V, Martienssen RA. 2003.
Understanding mechanisms of novel gene expression in polyploids. Trends
in Genetics 19: 141–147.
Ozias-Akins P, Roche D, Hanna WW. 1998. Tight clustering and
hemizygosity of apomixis-linked molecular markers in Pennisetum
squamulatum genetic control of apospory by a divergent locus that may
New Phytologist (2006) 171: 425–438 www.newphytologist.org © The Authors (2006). Journal compilation © New Phytologist (2006)
have no allelic form in sexual genotypes. Proceedings of the National
Academy of Sciences, USA 95: 5127–5132.
Quarin CL, Espinoza F, Martinez EJ, Pessino SC, Bovo OA. 2001. A rise
of ploidy level induces the expression of apomixis in Paspalum notatum.
Sexual Plant Reproduction 13: 243–249.
Ramsey J, Schemske DW. 1998. Pathways, mechanisms, and rates of
polyploid formation in flowering plants. Annual Review of Ecology and
Systematics 29: 467–501.
Richards A. 1986. Plant Breeding Systems. London: George Allen & Unwin.
Roche D, Hanna WW, Ozias-Akins P. 2001. Is supernumerary chromatin
involved in gametophytic apomixis of polyploid plants? Sexual Plant
Reproduction 13: 343–349.
Roy BA. 1995. The breeding systems of six species of Arabis (Brassicaceae).
American Journal of Botany 82: 869–877.
Schranz ME, Dobes C, Koch MA, Mitchell-Olds T. 2005. Sexual
reproduction, hybridization, apomixis, and polyploidization in the genus
Boechera (Brassicaceae). American Journal of Botany 92: 1797–1810.
Sharbel TF, Mitchell-Olds T. 2001. Recurrent polyploid origins and
chloroplast phylogeography in the Arabis holboellii complex (Brassicaceae).
Heredity 87: 59–68.
Sharbel TF, Voigt ML, Mitchell-Olds T, Kantama L, de Jong H. 2004. Is
the aneuploid chromosome in an apomictic Boechera holboellii a genuine
B chromosome? Cytogenetic and Genome Research 106: 173–183.
Sharbel TF, Mitchell-Olds T, Dobes C, Kantama L, de Jong H. 2005.
Biogeographic distribution of polyploidy and B chromosomes in the
apomictic Boechera holboellii complex. Cytogenetic and Genome Research
109: 283–292.
Sherwood RT, Berg CC, Young BA. 1994. Inheritance of apospory in
buffelgrass. Crop Science 34: 1490–1494.
Spillane C, Curtis MD, Grossniklaus U. 2004. Apomixis technology
development – virgin births in farmers’ fields? Nature Biotechnology 22:
687–691.
Stebbins G. 1950. Variation and Evolution in Higher Plants. New York:
Columbia University Press.
Taskin KM, Turgut K, Scott RJ. 2004. Apomictic development in Arabis
gunnisoniana. Israel Journal of Plant Sciences 52: 155–160.
Verduijn MH, Van Dijk PJ, Van Damme JMM. 2004. The role of
tetraploids in the sexual–asexual cycle in dandelions (Taraxacum). Heredity
93: 390–398.
West S, Lively C, Read A. 1999. A pluralist approach to sex and
recombination. Journal of Evolutionary Biology 12: 1003–1012.
de Wet J. 1968. Diploid–tetraploid–haploid cycles and the origin of
variability in Dichanthium agamospecies. Evolution 22: 394–397.