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Copy Number Variation in Transcriptionally Active Regions of
Sexual and Apomictic Boechera Demonstrates Independently
Derived Apomictic Lineages
W
Olawale M. Aliyu,a,1 Michael Seifert,b,c,d,1 José M. Corral,a Joerg Fuchs,e and Timothy F. Sharbela,2
a Apomixis
Research Group, Leibniz Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben, Germany
Inspection Research Group, Leibniz Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben, Germany
c Cellular Networks and Systems Biology, Biotechnology Center of the Technical University Dresden, D-01067 Dresden, Germany
d Innovative Methods of Computing, Center for Information Services and High Performance Computing, Technical University Dresden,
D-01067 Dresden, Germany
e Karyotype Evolution Research Group, Leibniz Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben, Germany
b Data
ORCID ID: 0000-0002-3450-2733 (T.F.S.).
In asexual (apomictic) plants, the absence of meiosis and sex is expected to lead to mutation accumulation. To compare
mutation accumulation in the transcribed genomic regions of sexual and apomictic plants, we performed a double-validated
analysis of copy number variation (CNV) on 10 biological replicates each of diploid sexual and diploid apomictic Boechera,
using a high-density (>700 K) custom microarray. The Boechera genome demonstrated higher levels of depleted CNV,
compared with enriched CNV, irrespective of reproductive mode. Genome-wide patterns of CNV revealed four divergent
lineages, three of which contain both sexual and apomictic genotypes. Hence genome-wide CNV reflects at least three
independent origins (i.e., expression) of apomixis from different sexual genetic backgrounds. CNV distributions for different
families of transposable elements were lineage specific, and the enrichment of LINE/L1 and long term repeat/Copia elements
in lineage 3 apomicts is consistent with sex and meiosis being mechanisms for purging genomic parasites. We hypothesize
that significant overrepresentation of specific gene ontology classes (e.g., pollen–pistil interaction) in apomicts implies that
gene enrichment could be an adaptive mechanism for genome stability in diploid apomicts by providing a polyploid-like
system for buffering the effects of deleterious mutations.
INTRODUCTION
Apomixis (asexual seed production in plants) derives from sexual seed production but circumvents meiosis and fertilization to
produce offspring genetically identical to the mother plant. Understanding the genetic mechanisms underpinning this switch is
of potential importance to agriculture and world food production, as it is considered a key enabling technology that could
facilitate the fixation of hybrid vigor (heterosis) in a single generation, thereby significantly reducing the costs associated with
hybrid seed production (Spillane et al. 2004). This naturally occurring form of seed production requires three important developmental steps: formation of an unreduced megaspore
(apomeiosis), development of an embryo without fertilization of
the egg cell (parthenogenesis), and endosperm formation with
(pseudogamy; Koltunow and Grossniklaus 2003) or without
(autonomous endosperm; Vinkenoog and Scott 2001) fertilization of the binucleate central cell. Apomictic plants differ with
respect to level of asexuality, ranging from obligate (i.e., producing
1 These
authors contributed equally to this work.
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Tim Sharbel ([email protected]).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.113.113860
2 Address
100% asexual seeds; Matzk et al. 2005) to facultative (i.e., capable of producing both sexual and apomictic seeds; Koltunow
1993; Matzk et al. 2005; Spillane et al. 2004).
Asexual reproduction has evolved independently and recurrently during angiosperm evolution (Van Dijk and Vijverberg
2005). Apparently, hybridization, polyploidy, and chromosomal
heteromorphy in both asexual plant and animal lineages (Roche
et al. 2001; Comai et al. 2003) could play important roles in the
induction, evolution, and/or maintenance of asexual reproduction.
For example, the genome-wide effects of hybridization and
polyploidy could be associated with deregulation of the original
sexual pathway to induce apomixis (Koltunow 1993; Carman
1997; Koltunow and Grossniklaus 2003; Sharbel et al. 2010).
Alternatively, they could contribute toward asexual lineage stability by buffering deleterious mutation accumulation, or via
heterosis-like effects (Richards 2003). As with sexual taxa, hybridization and polyploidy could additionally confer advantages
to apomicts through the creation of novel phenotypes, adaptation to diverse ecologies, and niche specialization (Adams 2007).
In plants, hybridization and polyploidy have been linked to
a number of phenomena on both the genomic and the transcriptomic levels, including genomic imbalance (Roche et al.
2001; Comai et al. 2003; Kantama et al. 2007), chromosomal
rearrangement and gene loss, interlocus concerted evolution of
ribosomal DNA repeats, unequal rates of sequence evolution in
duplicated genes, and changes in the DNA methylation and
gene expression (Adams 2007). In apomicts, the added fact that
The Plant Cell Preview, www.aspb.org ã 2013 American Society of Plant Biologists. All rights reserved.
1 of 16
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The Plant Cell
meiosis is absent or perturbed means that structural genomic
variation (i.e., mutation) arising via the above-mentioned processes has a lower probability of becoming purged, compared
with sexuals. In extending the idea of deleterious mutation accumulation in asexual genomes (i.e., Muller’s ratchet; Muller
1964; Neiman et al. 2010), such structural variation represents
a type of macromutation that should inevitably contribute toward extinction of asexual lineages (Muller 1964; Lynch and
Gabriel 1990; Charlesworth et al. 1993). Increased mutational
load in asexual taxa has, for the most part, been inferred through
the analysis of phenotypic or life history traits (Lynch 1985), but
on a higher level of organization it is clear that asexual taxa
exhibit relatively high levels of chromosomal instability (e.g.,
heteromorphy, aneuploidy, hemizygosity; Mayer and Aguilera
1990; Kantama et al. 2007; Akiyama et al. 2011). Considering
that these traits are ubiquitous among asexual taxa (Mayer and
Aguilera 1990; Roche et al. 2001; Kantama et al. 2007), it is
unclear whether they represent important factors associated
with the expression of asexuality (Kantama et al. 2007; Akiyama
et al. 2011), or whether they are the byproducts of disturbed
meiosis and have no evolutionary significance.
Analyses of the phenotypic effects of genetic variation have,
for the most part, focused on single nucleotide polymorphisms
(SNPs), but more recently copy number variants (CNVs) (i.e.,
large regions of the genome that differ in the copy number between individuals within a species because of duplication or
deletion events), as measured using array comparative genomic
hybridization (CGH), have emerged as an important source of
genetic and phenotypic variation in humans (Sebat et al. 2004;
Feuk et al. 2006; Beckmann et al. 2007; Conrad et al. 2010),
animals (Graubert et al. 2007; She et al. 2008; Cheeseman et al.
2009), and plants (Springer et al. 2009; DeBolt 2010; SwansonWagner et al. 2010). CNVs (Sebat et al. 2004) are DNA segments
in the base pair (bp) to megabase (Mb) range that vary in copy
number in comparison to a reference genome (Feuk et al. 2006).
These can be as simple as tandem duplication, or may involve
complex gain or loss of homologous sequences at multiple sites
(Redon et al. 2006). CNVs can arise from replication slippage,
nonallelic homologous recombination, nonhomologous end
joining, and retrotransposition (Conrad et al. 2010; Schrider and
Hahn 2010). Because CNV involves large segments of DNA,
they can significantly affect gene structure and dosage (Redon
et al. 2006; Henrichsen et al. 2009; Zhang et al. 2009), and, as
such, CNV has been described as a conditio sine qua non of
evolutionary divergence (Schrider and Hahn 2010) because it
contributes significantly to genetic diversity and evolution.
Moreover, CNV polymorphisms (i.e., duplications and deletions)
are known to be under different levels of natural selection
(Emerson et al. 2008; DeBolt 2010), with deletions being under
stronger purifying selection than duplications. The association
between CNV and diseases has been shown in humans and
animals (Aitman et al. 2006; Merikangas et al. 2009; Zhang et al.
2009; Beroukhim et al. 2010; Zhou et al. 2010), but fewer studies
have been done in sexual plants (Springer et al. 2009; DeBolt
2010; Swanson-Wagner et al. 2010).
The genus Boechera (Bocher’s rock cress; formerly Arabis), is
a wild relative of Arabidopsis thaliana and a model for studies of
population genetics, evolution, and apomixis. Boechera are
perennials and members of the Brassicaceae, distributed
throughout North America and Greenland (Koch et al. 1999;
Koch et al. 2003; Kiefer et al. 2009). Boechera has a basic
chromosome number x = 7 (Koch et al. 1999) and is characterized by diploid sexual, as well as diploid, aneuploid, and
polyploid (mostly 2n = 3x = 21) apomictic forms (Böcher 1951).
The relatively rare combination of apomixis and diploidy enables
comparisons of sexual and apomictic reproduction in the absence of concomitant ploidy variation. Apomictic Boechera are
characterized by Taraxacum-type diplospory whereby the
megaspore mother cell goes through meiosis I with restitution
nucleus formation (apomeiosis), followed by meiosis II leading to
dyads with nuclei of the same ploidy as the somatic cells of the
mother plant (Böcher 1951; Naumova et al. 2001). Apomictic
Boechera genotypes express both obligate and a strong bimodal expression of facultative apomixis (Schranz et al. 2005;
Aliyu et al. 2010), and there is evidence for CNV between sexuals and apomicts (Corral et al. 2009).
In this study, we have investigated genome-wide CNV in 20
different Boechera genotypes (10 sexual and 10 apomictic) using CGH to (1) compare mutation accumulation between sexuals
and asexuals in gene coding regions (Werren 2011), and (2)
identify reproduction-specific CNVs, which may reflect important loci for the evolution of apomixis in this genus. Although
CNV has been described as sequences that are present in different copy number in both reference and sample genomes
(Feuk et al. 2006; Springer et al. 2009), here we use the terms
duplications and deletions in the context of enrichment and
depletion (Grayson et al. 2010) relative to a pooled reference
sample (see Methods).
RESULTS
Genome-Wide CNV Is Reflective of At Least Three
Apomictic Lineages
Considering the distribution of normalized log ratios of CGH
measurements (Figure 1; see Supplemental Table 1 online,
Supplemental Figure 1 online), the vast majority of genomic
regions in apomictic and sexual genotypes (Table 1) were
characterized by log ratios with values of about zero, indicating
that these regions are not affected by CNV. In addition, the
distribution of log ratios showed a strong asymmetry toward the
negative tail (potentially depleted or polymorphic regions) rather
than the positive tail (potentially enriched regions; Figure 1; see
Supplemental Figure 1 online).
From 719,345 probes present on our tiling-array, our dye-swap
CGH analysis validated between 669,432 and 715,795 probes
per genotype (see Supplemental Table 2 online). Considering all
data together, a total of 313,568 probes showed copy number
polymorphism across the apomictic and sexual genomes, the
most significant of which were depletions (n = 304,593; 97%)
compared with enrichment (n = 8975 probes; 3%; Table 2).
There were differences between reproductive modes, with the
most probes (67%) showing CNV in the sexual genotypes,
compared with 33% in apomicts (Table 2; see Supplemental
Table 2 online).
CNV in Sexual and Apomictic Boechera
Figure 1. Distributions of log2 ratios of normalized CGH measurements
for 10 sexual (red) and 10 apomictic (blue) Boechera genotypes.
Hierarchical clustering of genomic regions across the 20
Boechera genotypes revealed four different lineages, in addition to
one outlier (B. schistacea Sex-9; Figure 2). The divergent position
of B. schistacea could be explained by its having the greatest
number of depletions (49,624; see Supplemental Table 2 online) in
addition to its geographic origin (Nevada). The four lineages can be
further differentiated into three lineages composed of both sexual
and apomictic genotypes (lineages 1, 3, and 4), and one lineage
composed exclusively of sexual B. stricta (lineage 2; Figure 2). The
three mixed lineages were characterized by apomictic genotypes representing different species and geographic origin:
lineage 1, B. polyantha from Idaho; lineage 3, B. polyantha,
B. retrofracta, B. divaricarpa, and B. stricta 3 B. retrofracta from
Montana; lineage 4, B. duchesnenses and B. lignifera from Utah
and B. divaricarpa from Montana (Figure 2, Table 1).
In considering each lineage separately, the distribution of
enrichment frequency appeared to differ between sexuals and
apomicts in lineage 3 only (Figure 3), although only a single
sexual individual was part of this comparison. On the other
hand, the range of depletion frequency per genotype followed
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a lineage-specific pattern and showed no apparent differences
between sexuals and apomicts (Figure 3). Finally, the distribution of enrichment to depletion frequency ratio (per genotype)
was broader for apomictic compared with sexual genotypes for
lineage 3 only (Figure 3).
The Boechera cDNAs corresponding to the 208,573 sexual
probes and 104,995 apomictic probes (Table 2) that demonstrated
significant CNV were physically mapped to the Arabidopsis genome, based upon homology between Boechera and Arabidopsis
cDNA sequences. In both cases, the probes were broadly dispersed with no apparent pattern across all five chromosomes of
A. thaliana (Figure 4). Notable is the absence of probes corresponding to the centromeric regions, in addition to the heterochromatic knob region of Arabidopsis chromosome 4 (i.e., gap to
left of position 2,655,006 on chromosome 4, Figure 4; [Borevitz
et al. 2007]).
Although sexual genotypes appeared to be characterized by
more extensive depletions compared with apomicts for all
Arabidopsis mappings (Figure 4), this was not the case if the
profiles were assigned to their respective lineages (Figure 2).
Close examination of the apomictic mappings demonstrated
qualitative similarities between samples from lineages 3 (Apo-1,
2, 3, 4, 7, and 8) and 4 (Apo-5, 6, and 10; Table 1, Figures 2 and
4). Furthermore, the sexual B. stricta (Sex-7, lineage 3) was
characterized by mappings that more closely resembled apomictic genotypes of the same lineage (Figure 4).
Negative Log Ratios Are Caused by CNV Depletions
and Not SNPs
Probes characterized by negative log2 ratios in the left tail of the
hybridization signal distributions are reflective of depletions in
the genotypes for the corresponding probes on the microarray
Table 1. Twenty Boechera Genotypes Used for CGH
IPK-Plant ID
Sample Name
B10-785
B10-797
B10-806
B10-817
B10-822
B10-865
B10-903
B10-829
B10-897
B10-881
B10-804
B10-833
B10-839
B10-913
B10-888
B10-902
B10-811
B10-851
B10-878
B10-791
Apo-1
Apo-2
Apo-3
Apo-4
Apo-5
Apo-6
Apo-7
Apo-8
Apo-9
Apo-10
Sex-1
Sex-2
Sex-3
Sex-4
Sex-5
Sex-6
Sex-7
Sex-8
Sex-9
Sex-10
a
ES Numbera
ES
ES
ES
ES
ES
ES
ES
514
517
753
805
524
776
765
ES
ES
ES
ES
ES
ES
ES
ES
552
555
910
773
791
503
661
760
Species
Reproductionb
Locality
U.S. State
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
Apomictic
Apomictic
Apomictic
Apomictic
Apomictic
Apomictic
Apomictic
Apomictic
Apomictic
Apomictic
Sexual
Sexual
Sexual
Sexual
Sexual
Sexual
Sexual
Sexual
Sexual
Sexual
Birch Creek
Ranch Creek
Mule Ranch
Vipond Park, Beaverhead
Vipond Park, Beaverhead
ES84/Map ID 74
Ranch Creek
Mule Ranch
Morgan Switch Back
ES88/Map ID 80
Bandy Ranch
Twin Saddle
Sagebrush Meadow
Parker Meadow
Wood Gulch-south
Trail Creek
Gold Creek
Golden Gate B
Toiyabe
Big Hole Pass
MT
MT
MT
MT
MT
UT
MT
MT
ID
UT
MT
ID
MT
ID
CO
ID
CO
CO
NV
MT
polyantha
retrofracta
stricta x retrofracta
divaricarpa
divaricarpa
lignifera
polyantha
divaricarpa
polyantha
duchesnensis
polyantha
stricta
stricta
polyantha
crandallii
polyantha
stricta
stricta
schistacea
polyantha
Refer to Schranz et al. 2005 for locality information. Blank cells indicate that the plant had no ES number from previous work.
Plant selected based on the flow cytometric seed screening analysis of $300 single seeds per accession (Aliyu et al. 2010).
b
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The Plant Cell
Table 2. Number of Probes Showing CNV in Apomictic and Sexual
Boechera Genotypes
Mode of Reproduction
CNV
Apomictic
Sexual
Depletions
Enrichment
All probes showing CNV
99,795 (32.76%)
5,200 (57.94%)
104,995 (33.48%)
204,798 (67.24%)
3,775 (42.06%)
208,573 (66.52%)
(Figure 1; see Supplemental Figure 1 online). Alternatively, relatively low hybridization signal on the array could have resulted
from SNP differences between the sample and probe sequences,
which hindered hybridization between them (Swanson-Wagner
et al. 2010).
We therefore tested whether the cDNAs used to design the
microarray probes were characterized by relatively high levels of
SNPs in sexuals versus apomicts, by individually mapping three
sexual and three apomictic cDNA libraries (sequenced using 454
FLX technology; Sharbel et al. 2009, 2010) to the contig sequences used to design the microarray. With the use of CLC
Genomics workbench, SNP numbers were compared against
the six cDNA libraries at both 103 and 43 coverage thresholds.
At the 103 coverage level, a total of 873 SNPs were found, 298
(34%) and 575 (66%) of which were found in the sexual and
apomictic libraries, respectively. Similarly at the 43 coverage
level, of a total of 5036 SNPs, 1685 (33%) and 3371 (67%) were
identified in the sexual and apomictic libraries, respectively.
Assuming both alleles of most genes are equally expressed in
both sexuals and apomicts, a higher number of SNPs in the
apomictic cDNA leads to the expectation of discovering more
false depletions in apomicts attributable to hybridization artifacts arising through differences between probe and test sequences. The higher number of apomictic SNPs attests to their
hybrid nature and contradicts higher levels of depletions observed in sexuals. Hence these SNP data, although not complete in terms of coverage of the Boechera genome, support
CNV in the form of depletions rather than SNP polymorphisms
as the explanation for probes showing negative log2 ratios
(Figure 1; see Supplemental Figure 1 online).
Genomic Regions Differentiating Sexual from
Apomictic Boechera
In an attempt to identify CNV patterns that were specific to either
sex or apomixis, we could not identify any array probes that
were consistently different (e.g., enriched or depleted) between
all sexual and apomictic genotypes. We thus used a relaxed
threshold to identify sex- or apomixis-specific genomic markers
that were best able to discriminate between sexual and apomictic genotypes; a genomic marker was considered discriminative between sexual and apomictic genotypes only if this
marker was identified at least eight times (i.e., 8 of 10 biological
replicates used in the analysis) as depleted, unchanged, or enriched across all sexual genotypes and did not have more than
twice the same status across all apomictic genotypes, or vice
versa, respectively. Using these criteria, we identified 110 array
probes that showed a tendency of progressive reduction in
accumulation of depletions (deep red) to unchanged (orange)
and enriched (deep yellow), as observed from sexual to apomictic
Figure 2. Hierarchical clustering of 10 sexual and 10 apomictic Boechera genotypes (Table 1) based on decoded underlying copy number states
(coded as depletion 21, unchanged 0, enrichment +1) assigned to all cDNA microarray probes (see Methods). Bootstrap values are placed at each node
of the tree. Numbers in circles show four differentiated lineages defined by genome-wide CNV.
CNV in Sexual and Apomictic Boechera
5 of 16
genotypes (Figure 5). We note in addition that the identified set
of genomic markers had the greatest discriminative power to
distinguish between sexual and apomictic genotypes with respect to our selection criterion. Moreover, considering that we
examined 720K probes, the vast majority of which did not
demonstrate reproduction-specific CNV, it is highly unlikely that
a random selection of 110 array probes could have led to
a similar result.
An analysis of the Boechera cDNAs corresponding to the
110 probe sequences demonstrated that in many cases two or
more of the 110 probes mapped to a single cDNA, thus
demonstrating technical replication (i.e., more than one probe
per gene) support for the identification of sex-specific CNV.
The 110 array probes thus corresponded to 64 cDNAs for
which a gene ontology (GO) analysis was performed. Of these,
31 (48%) could be annotated, and, of these, 13 (42%) could
be attributed to transposable elements (TEs) (Figure 5; see
Supplemental Table 3 online).
Overall, a GO analysis for overrepresentation of particular
classes of genes showing CNV demonstrated a similar pattern
of significant classes between sexuals and apomicts (see
Supplemental Figure 2 online). Most of the genes characterized
by depletions in both reproductive classes were involved in
regulatory, transporter, or catalytic activity, although the number
of significant overrepresentation classes was higher in sexuals
(see Supplemental Figures 2A and 2Bonline), The pattern of
significantly overrepresented GO classes of enriched genes was
similar between sexual and apomictic genotypes, although the
level of significance for most overrepresented and shared GO
classes was frequently higher in apomicts (see Supplemental
Figures 2C and 2D online).
Interestingly, pollen–pistil interactions (GO:0009875) were overrepresented for enriched genes in both sexuals and apomicts,
although to a higher significance level in apomicts (see
Supplemental Figures 2C and 2D online). Together, 16 Arabidopsis
genes characterized by pollen–pistil interactions GO terms
corresponded to these enriched Boechera coding regions, eight
of which were shared between both reproductive forms (see
Supplemental Table 4 online). Three of the 16 genes encoded
proteins with ATP binding properties, whereas the remaining
genes encoded proteins characterized as S-locus lectin protein
kinases (see Supplemental Table 4 online).
Six genes showing homology to the microarray probes characterized by apo-specific CNV were found across all A. thaliana
chromosomes, whereas those (many more) showing sex-specific
CNV appeared to be physically linked to one another in a number of clusters (see Supplemental Figure 3 online).
Lineage-Specific Patterns in Genome Size and
TE Frequencies
A flow cytometric analysis of absolute genome size (pg/2C) for
all genotypes was performed to examine the relationship between
Figure 3. Frequency distribution of (A) enrichment, (B) depletions, and
(C) enrichment/depletions (per genotype) derived from CGH data for 10
apomictic (dark gray, left boxplot) and 10 sexual (light gray, right
boxplot) Boechera genotypes from four lineages. Note that only a single
apomictic genotype was present in lineage 1, only a single sexual genotype was found each in lineages 3 and 4, and no apomictic genotypes
were found in lineage 2 (Table 1; Fig. 2).
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The Plant Cell
Figure 4. Physical distribution of Boechera CNV mapped to A. thaliana chromosomes 1 to 5 in 10 each (A) apomictic (Table 1) and (B) sexual
genotypes (Table 1). For each genotype, the positions of depletions and enrichment are indicated as gray bars below and above the vertical midpoint,
respectively, of each chromosome.
CNV and genome size diversity. The 2C genome size of sexuals
ranged from 210 Mbp (0.429 pg/2C) in B. polyantha to 247 Mbp
(0.504 pg/2C) in B. schistacea, whereas for apomicts, genome
size ranged from 221 Mbp (0.451 pg/2C) in B. polyantha to 247
Mbp (0.505 pg/2C) in B. retrofracta (Table 3). Significant intraspecific variation in genome size was also apparent for sexual
B. polyantha and B. stricta, and for apomictic B. polyantha and B.
divaricarpa (Figure 6A). No significant correlations between CNV
per genotype and genome size were found (coefficient of determination: enrichment Apo R2 = 0.309; enrichment Sex R2 = 0.314;
depletion Apo R2 = 0.104; depletion Sex R2 = 0.0.096). Of note,
evidence was found for lineage-specific genome size, in addition to a trend for larger genome sizes in apomicts from lineages
1 and 4 (Figure 6B).
To find homologs to Arabidopsis TEs (The Arabidopsis Information Resource [TAIR]10 TE), a BLAST analysis of Boechera
cDNA sequences of array probes demonstrating significant CNV
showed that TEs of all families have undergone higher levels of
depletion compared with enrichment in both sexual and apomictic
Boechera genotypes from all lineages (Figure 7). Furthermore, the
CNV in Sexual and Apomictic Boechera
7 of 16
Figure 5. Heatmap representing copy number states of microarray probes identified as being best discriminative between sexual and apomictic
genotypes (Table 1). The heatmap was computed based on the average log ratio of corresponding probes measured in both replicates (i.e., dye swap)
of each genotype. Color codings representing log ratios reflective of depletions (deep red) and enrichment (deep yellow), and proportions (green lines) of
probes corresponding to the log ratios are shown in the subfigure in the left upper corner.
frequency of depletion pattern across the different TE families
was similar in the different lineages, with the highest being for
long term repeat/Copia elements in all cases (Figure 7). In
contrast, the pattern of enrichment was qualitatively different
across the different lineages (Figure 7). Finally, different families
of TE appeared to be enriched in all apomictic genotypes in
lineage 3, although the comparison involved only one sexual
genotype.
Although the measurement scale differs between the array (log2
ratio fluorescence) and qRT-PCR (quantification relative to
a single copy locus) data, the pattern of CNV for both sexual and
apomictic genotypes was strikingly similar for both analyses in
;80% of the loci tested (70 of the 88 profiles; see Supplemental
Figure 4 online).
DISCUSSION
Highly Consistent Validation Patterns between Dye-Swap
CGH and Quantitative RT-PCR Data
Besides the standard dye-swap CGH design, we performed an
additional quantitative RT-PCR (qRT-PCR) validation using
primers designed from 22 genes that expressed significant
CNV between two sexual (Sex-3 and Sex-6; see Supplemental
Table 1 online) and two apomictic (Apo-1 and Apo-3; see
Supplemental Table 1 online) genotypes from the CGH analysis.
Insertion–deletions and SNPs have typically been the focus of
diversity studies, but advances in microarray technology have
led to a growing interest in the influence of CNV on phenotypic
variation (Sebat et al. 2004; Beckmann et al. 2007; Springer
et al. 2009; DeBolt 2010). Here, we have performed a doublevalidated (i.e., both dye-swap and qRT-PCR validation) analysis
of CNV on 10 biological replicates each of diploid sexual and
diploid apomictic Boechera genotypes using a high-density
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The Plant Cell
Table 3. Genome Size (pg/2C and Mbp/1C) for Sexual and Apomictic Boechera Genotypes (Table 1)
Sample Name
Species
Mean (pg/2C)
Apo-1
Apo-2
Apo-3
Apo-4
Apo-5
Apo-6
Apo-7
Apo-8
Apo-9
Apo-10
Sex-1
Sex-2
Sex-3
Sex-4
Sex-5
Sex-6
Sex-7
Sex-8
Sex-9
Sex-10
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
0,498
0,505
0,469
0,458
0,461
0,462
0,462
0,480
0,451
0,488
0,464
0,484
0,467
0,436
0,452
0,438
0,478
0,480
0,504
0,429
a
polyantha
retrofracta
stricta x retrofracta
divaricarpa
divaricarpa
lignifera
polyantha
divaricarpa
polyantha
duchesnensis
polyantha
stricta
stricta
polyantha
crandallii
polyantha
stricta
stricta
schistacea
polyantha
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
0,008
0,011
0,005
0,007
0,004
0,010
0,005
0,008
0,008
0,004
0,006
0,014
0,009
0,012
0,007
0,009
0,010
0,008
0,011
0,011
Minimum (pg/2C)
Maximum (pg/2C)
Significancea
0.483
0.493
0.462
0.449
0.455
0.452
0.455
0.470
0.440
0.484
0.455
0.454
0.450
0.420
0.442
0.427
0.463
0.469
0.488
0.414
0.508
0.524
0.474
0.467
0.467
0.483
0.471
0.496
0.464
0.496
0.473
0.497
0.479
0.450
0.462
0.452
0.490
0.489
0.516
0.447
h, i
i
d, e, f
c, d
c, d
c, d
c, d
f, g
b, c
g, h
c, d, e
g, h
d, e, f
a
b, c
a, b
e, f, g
f, g
i
a
Genotypes with different letters have significantly different means (P < 0.05; calculated using Holm–Sidak pairwise multiple comparison procedure).
(>700K) custom microarray, with the goal of comparing mutation
accumulation and CNV variation between the transcribing regions of their genomes. We opted for an array CGH approach
rather than one based upon RNA sequencing technology because (1) no genome sequence backbone upon which sequence
data could be assembled is available for Boechera; (2) even if
such a genome backbone were available, apomicts have characteristic large-scale chromosomal rearrangements (Kantama
et al. 2007), which would both complicate and obscure assembly patterns; and (3) although sequencing costs are steadily
decreasing, the costs of an RNA sequencing experiment on the
scale of analysis presented here (i.e., large numbers of biological
replicates) remain significantly higher than array CGH.
Of 313,568 array probes that showed CNV, ;97% (304,593,
Table 2) were depletions. The low frequencies of enrichment for
all Boechera genotypes, irrespective of reproductive mode
(Table 2, Figure 3), strongly suggest that their accumulation is
counterselected (Charlesworth and Charlesworth 1983; Le
Rouzic et al. 2007). As cDNA was used to design the custom
CGH microarray used here, our CNV analyses are biased toward
coding genes, which, compared with noncoding regions, are
likely under higher levels of selection pressure (Mills et al. 2011).
This situation could explain the lack of significant correlations
between genome size and CNV (Figure 6). Finally, as no general
signal in terms of sex- or apomixis-specific CNV could be
identified here, this conclusion can be applied only to transcribing
regions of the genome, and possibly reflects a limitation of our
approach. In the same light, apomictic lineages represent independently evolving entities characterized by disturbed meiosis
and hybridization, which together could potentially hinder the
maintenance of apomixis-specific CNV, if any initially existed.
While the qRT-PCR validations of the array were highly consistent, we suspect that the relatively few inconsistencies (e.g., 18
of 88 profiles; see Supplemental Figure 4 online) could have
arisen through qRT-PCR primer design over unpredicted cDNA
splicing sites, or differences in amplification efficiency caused
by target sequence variability between the species/genotypes
used in the microarray and the ones used for the cDNA library.
CNV Demonstrates Multiple Evolutionary Origins of
Asexual Lineages
In the long term, deleterious mutation accumulation in an
asexual lineage should eventually lead to its extinction (i.e.,
Muller’s ratchet; Felsenstein 1974; Neiman et al. 2010). In small
obligate asexual populations, this process is hypothesized to
accelerate (i.e., mutational meltdown; Lynch et al. 1993), and
this has been proposed as the underlying reason for the rarity of
obligate asexuality. Apomictic Boechera challenge these hypotheses because (1) obligate apomixis has been identified in
both diploids and triploids (Aliyu et al. 2010), (2) apomictic
genotypes are genetically variable and geographically widespread (Kiefer et al. 2009), and (3) apomicts are characterized by
phenotypic (Voigt-Zielinski et al. 2012) and chromosomal
(Kantama et al. 2007) variation that is reflective of deleterious
mutation accumulation. Thus multiple origins of apomictic lineages from different sexual ancestors through time are a likely
explanation for their long-term evolutionary success, despite
Muller’s ratchet.
The multiple-origin hypothesis in no way implies the de novo
convergent and repetitive evolution of apomixis factors via mutational processes; rather it implies either (1) convergent patterns
of apomixis-specific gene expression (i.e., deregulation) triggered
by frequent hybridization between different sexual lineages
(Sharbel et al., 2010), or (2) the spread of singly derived apomixis
factors via sex (e.g., pollen) into different sexual backgrounds
CNV in Sexual and Apomictic Boechera
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Figure 6. Genome size variation. (A) Boxplots showing technical replicate distribution of genome size for sexual and apomictic Boechera genotypes.
(B) Genome size variation per lineage of sexual (light gray) and apomictic (dark gray) Boechera (Figure 2).
(Lovell et al., 2013). Although strong evidence exists for multiple
origins of apomictic lineages via hybridization between sexual
Boechera species (Koch et al. 2003; Dobeš et al. 2004a, 2004b;
Kantama et al. 2007; Beck et al. 2012), so far no attempt has
been made to understand the number of origins or the relative
evolutionary ages of these lineages.
Here we have shown, based upon genome-wide patterns of
depletion and enrichment, that five lineages (i.e., four clusters
and the divergent B. schistaceae-Sex-9 sample) can be identified
from the genotypes selected for this experiment (Figure 2). Three
of the clusters (lineages 1, 3, and 4) are characterized by both
sexual and apomictic genotypes (Figure 2), and considering that
apomixis is derived from sexuality in Boechera, these data are
consistent with at least three independent origins of apomixis
(for the samples analyzed here) from different sexual backgrounds. In support of this, some taxa grouped here represent
divergent internal transcribed spacer genotypes (e.g., B. crandallii
and B. lignifera in lineage 4; Kiefer and Koch (2012). Furthermore,
10 of 16
The Plant Cell
Figure 7. TE frequencies. Average frequencies of depletions (A) and enrichment (B) for TE families in sexual (light gray) and apomictic (dark gray)
Boechera genotypes across four lineages. Note that lineage 2 contains no apomictic genotypes. Error bars show 23 the standard error in cases in
which the data from two or more genotypes were averaged per lineage (Table 1 and Figure 3).
CNV in Sexual and Apomictic Boechera
11 of 16
as genome-wide CNV patterns are similar for both sexual and
apomictic genotypes within each lineage (Figure 4), it appears that
the derived apomictic genotypes have adopted the genomic
(CNV) characteristics of their sexual ancestor. The spectrum of
enrichment and depletion frequencies for apomicts and sexuals
within lineages additionally supports the idea (albeit with small
sample sizes) that each apomixis origin reflects a snapshot of the
sexual ancestor from which it originated (Figure 3). Finally, genome size demonstrated a trend of within lineage similarity rather
than taxon specificity, thus further supporting the idea that phylogenetic history was the underlying factor responsible for patterns in CNV and genome size (Figure 6).
One aspect of this hypothesis is that the lineages analyzed
here could be of differing evolutionary age. In this light, lineage
4, which is composed of four different sexual and apomictic
species from Utah, Colorado, and Montana, is a potential candidate for a relatively old lineage, which is reflected in the large
phylogenetic and geographic distance encompassed in the
group (Figure 2). Finally, considering the level of genetic (chloroplast DNA, microsatellites, CNV and so on), biogeographic,
and taxonomic diversity represented by the genotypes examined here, we thus favor the single origin and spread of apomixis
factors (Lovell et al., 2013) over convergent patterns of gene
expression through hybridization as the trigger for apomixis in
Boechera.
tendencies for depletions of TEs (Figure 5; see Supplemental
Table 3 online).
The data presented here show no general differences in TE
copy number, for any TE family, between sexual and apomictic
genotypes, except for lineage 3 (Figure 7). Interestingly, although the pattern of depletion across different TE families for
both sexuals and apomicts was similar in all lineages, apomicts
from lineage 3 showed (1) enrichment in LINE/L1 and long term
repeat/Copia elements (compared with other lineages) that was
(2) higher than what was found in the (albeit single) sexual
sample (Figure 7). As the apomicts from lineage 3 also showed
great variability in frequencies of enrichment for non-TE loci
between one another (Figure 3), the similar divergent pattern of
TE family enrichment (compared with other lineages) shared by
lineage 3 apomicts is consistent with TE accumulation.
Finally, we point out that as the custom microarrays used here
were designed using flower-specific cDNA (Sharbel et al. 2009;
2010), the TE sequences on the array were derived from active
elements that hypothetically have not lost their identity through
DNA sequence erosion through time. Hence, the identification of
apparent enrichment for different families of TE in lineage 3
apomicts is (1) a conservative estimate and (2) suggestive of
a relatively recent and active mechanism. In support of this idea,
13 of 31 probes showing apomixis- and sex-specific CNV were
TE related (Figure 5; see Supplemental Table 3 online).
Genomic Parasite Frequencies Reflect Both Phylogenetic
History and Reproductive Mode
CNV as an Adaptive Mechanism in Apomictic Boechera
Considering their ability to self-propagate and their potentially
deleterious effects on gene expression and chromosome rearrangements through ectopic recombination (Petrov et al. 2003),
TEs are genomic parasites. Because of their deleterious effects,
TE insertions are expected to be under purifying selection
(Hollister and Gaut 2009), leading to lower levels of TEs in selfers
compared with outcrossers (Morgan 2001; Lockton and Gaut
2010). Moreover, the mating system in plants has been shown to
influence TE accumulation and distribution (Morgan 2001;
Lockton and Gaut 2010), and there is evidence that genome size
variability is influenced by TEs (Bennetzen et al. 2005; Ray and
Batzer 2011). In apomicts, TE accumulation has been implicated
in the evolution of genomic regions that harbor apomixis factors
(Akiyama et al. 2004; Okada et al. 2011) and is hypothesized to
be a factor that contributes to eventual lineage extinction (Dolgin
and Charlesworth 2006).
A number of factors could lead to differential purifying selection on CNV between sexual and apomictic Boechera: (1)
Sexuals are highly selfing (Song et al. 2006), and (2) apomicts
are hybrid in origin (Koch et al. 2003; Dobeš et al. 2004a, 2004b;
Beck et al. 2012) and have disturbed or absent meiosis (apomeiosis;
Böcher 1951; Kantama et al. 2007). Thus the probability of
recombination and syngamy leading to loci that are homozygous for deleterious recessive alleles would be higher in sexual
than in apomictic plants, and this would apply to both TE insertions and other types of mutation. Hence, assuming a negative fitness effect on such homozygous individuals, followed
by purifying selection, the overall effect would be lower mutation
(and TE) levels in sexuals, and this is reflected in sexual-specific
Polyploidy is highly characteristic of asexual plants and animals,
an association that has been explained from a number of perspectives, including allelic dosage and control, escape from
sterility, and heterosis-like effects (Mogie 1986; Asker and
Jerling 1992). This effect is reflected in comparisons between
diploid and triploid apomictic Boechera genotypes, which show
a buffering effect of polyploidy on reproductive trait variance
(Voigt-Zielinski et al. 2012).
In the same light, in diploid apomictic Boechera genotypes,
gene duplication (enrichment) may provide a polyploidy-like
mechanism whereby the effects of deleterious mutations are
buffered by extra allele copies (Tanaka et al. 2009). In support of
this concept, GO analyses of enriched (duplicated) genes
demonstrate differences between apomicts and sexuals. For
example, genes associated with pollen–pistil interactions are
more significantly overrepresented in apomicts compared with
sexuals (see Supplemental Figures 2C and 2D online;
Supplemental Table 4 online). Considering that fertilization of the
central cell, regardless of parthenogenetic development of the
apomeiotic egg cell, is highly conserved among diploid apomictic Boechera genotypes (Aliyu et al. 2010), maintenance of
gene duplicates for pollination could conceivably be advantageous for proper endosperm genome balance.
Duplications could also provide the fodder for adaptation,
irrespective of reproductive mode. Machado and Renn
(Machado and Renn 2010) suggested gene duplication as an
important source of functional novelty, as it has been shown to
be involved in adaptive evolution in response to diet (Zhang
et al. 2002), chemical challenge (Labbé et al. 2007), and reproductive incompatibility (Lynch and Force 2000). Our GO
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The Plant Cell
analyses indeed showed duplications for genes involved in both
abiotic and biotic stress responses (see Supplemental Figures
2C and 2D online), potentially providing the genetic variability to
rapidly respond to environmental factors in both sexual and
apomictic forms.
Conclusion
We have shown that CNV for transcriptionally active genomic
regions is prevalent in both sexual and apomictic Boechera, and
that the genome-wide pattern of CNV is reflective of phylogeographic history rather than reproductive mode. Importantly,
similar patterns of genome-wide CNV shared between sexual
and apomicts in divergent lineages provide strong support for
independent origins of apomixis from different sexual backgrounds and is furthermore consistent with the sexual spread of
apomixis-specific factors as the apomixis induction mechanism.
Some evidence for mutation accumulation (i.e., Muller’s ratchet)
was provided by differences in TE CNV frequency between
sexuals and apomicts, in addition to genome size variation in
lineage 4, although our ability to measure such differences was
likely hindered by the use of a CGH array designed from gene
coding regions, which, compared with noncoding regions, are
likely under higher levels of selection pressure in both sexuals
and apomicts.
METHODS
Plant Materials
According to recent nomenclature, the genus Boechera is composed of
71 sexual diploid species and 38 hybrid taxa with variable ploidy (AlShehbaz and Windham 2006, 2007a, 2007b). Based on a previous largescale flow cytometric seed screening analysis of 71 Boechera genotypes
from 14 different taxa (Aliyu et al. 2010), 20 diploid genotypes with a high
penetrance (i.e., high-facultative; Aliyu et al. 2010) of apomeiosis (n = 10)
and sexuality (n = 10) were selected for the CGH analysis (Table 1).
Flow Cytometric Seed Screening
The penetrance of sexuality and apomixis in all genotypes was measured
using a combination of high throughput (see Aliyu et al. 2010) and
conventional razor-chopping (Matzk et al. 2000) flow cytometric seed
screening methods. Isolated nuclei from ;60 single seeds ($48 single
seeds for the high throughput procedure, and 12 single seeds for razorchopping) were analyzed per plant. Seeds were sampled from at least five
plants per genotype and analyzed. Seeds from the diploid sexual B. stricta
(SAD12; Schranz et al. 2005) were used as an external reference standard
throughout the measurements, as it produced seeds with exclusively 2C
embryo to 3C endosperm ratios, reflecting a diploid embryo composition of C (where C denotes the monoploid DNA content) maternal (Cm)
+ C paternal (Cp) = 2C genomes, and an endosperm composition of
2Cm + Cp = 3C. Reproductive pathways were constructed based on the
relationship between embryo and endosperm ploidy peaks from the
cytometric histogram profiles (Matzk et al. 2001). All sexual plants were
characterized by 2C:3C seeds (i.e., derived through reduced female
gametes and fertilization by reduced male gametes), and all apomictic
plants were characterized by predominant 2C:6C seed production (i.e.,
derived from autonomous development of female unreduced egg cells
[2x] with pseudogamous fertilization of female unreduced central cells [4x]
by unreduced male gametes [2x]; Aliyu et al. 2010).
Genome Size Analysis
Absolute genome size of the 20 Boechera genotypes used for CGH
analysis (Table 1) was measured flow cytometrically. Approximately 40
mg of young leaf tissue of each plant was processed together with an
internal reference standard (leaf tissue of Arabidopsis thaliana, var Columbia-0 [Col-0], with a genome size of 157 Mbp corresponding to 0.16
pg/1C; Bennett et al. 2003) in a petri dish containing 0.8 mL cold nuclei
isolation buffer (Galbraith et al. 1983) supplemented with DNase-free
RNase (50 µg/mL) and propidium iodide (50 µg/mL). The nuclear suspension was filtered through a 35-µm mesh filter and incubated for at least
10 min on ice before analysis. The relative fluorescence intensities of
isolated nuclei were measured with a FACStarPLUS (BD Biosciences) flow
sorter equipped with an argon ion laser INNOVA 90C (Coherent) aligned to
514 nm, and 9000 to 10,000 nuclei per sample were analyzed using the
software CellQuest 3.3 (BD Biosciences). At least four plants were analyzed per genotype in two replicates, except for B10-913, B10-878, and
B10-881, for which only three and two plants, respectively, were available.
Replicate measurements were further performed on different days
(Dolezel and Bartoš 2005). Absolute genome size was computed based
on the histogram G1 peak mean of sample (Boechera)/histogram G1 peak
mean of reference (Arabidopsis) multiplied by the genome size of the
reference (Arabidopsis). Genome size was then converted to base pair
(Mb/1 pg) using a factor of 1 pg = 978 Mb (Dolezel et al. 2003).
Microarray-Based CGH
A custom 3 3 720K Boechera-specific microarray (Roche NimbleGen)
was designed from data generated from six flower-specific cDNA libraries
sequenced using 454 FLX technology (see Sharbel et al. 2009; 2010).
Genomic DNA (gDNA) was extracted from the leaf tissue of a single plant
of each of the 20 genotypes (Table 1), using a modified cetyltrimethyl
ammonium bromide method described by Mace et al. (2003). DNA quality
was determined on a 0.7% agarose gel and with a NanoDrop 3300
Fluorospectrometer (Thermo Scientific, Wilmington, DE; http://www.
nanodrop.com/). To ensure optimal DNA yield after labeling, DNA with
concentrations between 450 ng/mL and 1.0 mg/mL and absorbance
readings of A260/A280 $ 1.8 and A260/A230 $ 1.9 (recommended by Roche
NimbleGen) were used for the labeling procedure. A total of 1.0 µg of
gDNA per sample was used for each labeling reaction and hybridization
thereafter.
A dye-swap experimental design was used for technical replication, in
which both test and reference samples are interchangeably labeled with
the two dyes (Cy3 and Cy5; Roche NimbleGen) to minimize error caused
by dye bias. A pooled reference sample was used, whereby an equal
concentration of gDNA from each of the 20 genotypes was combined as
a reference in each labeling reaction and hybridization (Scherer et al. 2007;
Ju et al. 2010). The idea is to encompass as much variation as possible
such that CNV is averaged on the array, leading to the identification of
individual signals that will stand out significantly against the pool (Scherer
et al. 2007; Ju et al. 2010). Samples were randomized across the three
subarrays of each 3 3 720K array such that two samples from the same
reproductive mode were never placed together on the same slide during
hybridization.
After hybridization, washing, and drying (following the manufacturer’s
protocols; Roche NimbleGen) the arrays were scanned at 2 µm doublepass resolution with an Agilent Microarray G2565BA Fluorescent Scanner
(Agilent Technologies). Each 3-plex image (3 3 720K) was split into three
separate arrays and aligned for data extraction using the NimbleScan
software (version 2.6; Roche NimbleGen). The NimbleScan software
performs spatial correction of the data, thereby reducing artifacts and
noise before applying q-spline fit normalization (Workman et al. 2002).
For the dye-swap experimental design, two CGH profiles (Cy3 and Cy5)
were obtained, each representing the log2 ratios of measured fluorescence
CNV in Sexual and Apomictic Boechera
intensities for the test (sexual or apomictic genotype; Table 1) versus reference sample (mixture of different genotypes, see above) for all 719,346
probes on the array (720K minus control probes). Genome-wide CGH
profiles of each genotype were normalized by applying quantile normalization (Bolstad et al. 2003) to both CGH profiles from the dye swap.
All normalized CGH profiles were analyzed by a mixture model of three
Gaussian distributions described in Seifert et al. (2012) to identify depletions, enrichment, and unchanged regions between a test genotype
and the reference sample by specifically adapting the parameters of the
mixture model to both CGH profiles (replicates from each dye swap; see
Supplemental Figure 1 online) of each test genotype using a Bayesian
Expectation Maximization algorithm. Identification of depleted, unchanged, and enriched regions was done by performing a decoding of the
measured log2 ratios into the most likely underlying state of each probe
(depleted, unchanged, or enriched). Only probes that were identified as
depleted or enriched in both CGH profiles (dye-swap replicates) of a test
sample (genotype) were considered as being depleted or enriched in this
genotype, while all other probes were considered as being unchanged
(i.e., no CNV variation with respect to the pooled reference). This resulted
in a reproducible set of genomic regions affected by depletions or enrichment for each genotype.
We additionally note that the interpretation of the decoding status
(depleted, unchanged, enriched) of some genomic regions may be biased
owing to the usage of a pooled reference, which was used, as no
complete high-quality genome sequence is available for any of the analyzed Boechera genotypes. Following similar studies (Scherer et al.
2007; Ju et al. 2010), the usage of a pooled reference enables the
quantification of genotype-specific CNV that stand out significantly
against the pool. Our general observation that the vast majority of analyzed genomic regions do not differ between the Boechera genotypes is
also consistent with similar findings in other plants (e.g., Clark et al. 2007;
Springer et al. 2009).
Hierarchical Clustering of Boechera Genotypes
Hierarchical clustering of the Boechera genotypes was performed based
on the classification of genomic regions in individual CGH profiles provided by the mixture model (see previous section). Genomic regions were
identified as depleted, unchanged, and enriched (coded to 21, 0, and +1
respectively), and distances between each pair of genotypes were calculated by the sum of the absolute differences among the corresponding
pairwise numerical decoding of genomic regions. The resulting distance
matrix was considered for hierarchical clustering using the Pvclust
package (Suzuki and Shimodaira 2006) by performing a standard Ward
clustering, which minimizes the total within-cluster variance, in combination with bootstrapping to analyze the significance of identified clusters.
Computed approximately unbiased probability values were considered
for evaluating the significance of clusters, whereby values close to 100
support a cluster’s potential biological relevance.
GO Analysis
Array probes that demonstrated significant CNV were traced back to their
original Boechera cDNA sequences (based upon assemblies of 454 reads;
Sharbel et al. 2009; 2010) and the corresponding full-length sequences
were used in a BLAST search to find Arabidopsis cDNA homologs
(TAIR10) using the following parameters: blastall –p blastn -m8 -e 1e-3
-W7 -r1 –q -1 -i (Altschul et al. 1997). Corresponding TAIR10 hits were
then used for a GO analysis using AgriGO (http://bioinfo.cau.edu.cn/
agriGO/index.php) for GO term enrichment, using the TAIR database as
background comparison set, with the following analysis parameters:
Fisher test, Yekutieli (FDR under dependency) adjustment for multiple
tests, significance level 0.01, minimum number of mapping entries = 5,
and plant GO slim GO.
13 of 16
The same Boechera cDNAs were used in a BLAST search to find
homologs to Arabidopsis TEs (TAIR10 TE) using the following parameters:
blastall -p blastn -m 8 -e 1e-3 -W 7 -r 1 -q -1 –i (Altschul et al. 1997). TE
homologs of cDNAs corresponding to array probes that showed significant CNV were selected using an E-value threshold of 2 3 10211.
qRT-PCR Validation of Genes Demonstrating CNV
Primers were designed for 22 genes that showed significant CNV (see
Supplemental Table 1 online). A total of 20 ng of genomic DNA was
isolated from four (two sexual and two apomictic genotypes used in the
CGH analysis; see Supplemental Table 1 online) genotypes and amplified
with SYBR Green 23 PCR Master Mix using a 7900HT Real-Teal PCR
Systems (all from Applied Biosystems) in a 10-mL reaction volume. Mean
cycle threshold value was calculated based on three technical replicates
per biological sample. The relative copy number was determined by the
Delta-Delta Mean Cycle Threshold method by comparison to a genomic
control known to be present in one copy in each genome (Yuan et al. 2007;
Springer et al. 2009).
Data Access
All microarray data used in this article have been submitted to the NCBI
GEO (Gene Expression Omnibus) data repository under the following
accession numbers: GSE51596.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Histograms of log2 Values Showing Normal
Distribution of Extracted CGH-CNV Data from the 40 Microarrays
Analyzed for 10 Apomictic and 10 Sexual Boechera Genotypes.
Supplemental Figure 2. GO Analysis Showing Key Biological Processes and Molecular Functions of All Significant CNV Sequences in
Boechera Genotypes.
Supplemental Figure 3. Physical Location of A. thaliana Homologs to
Boechera cDNAs Corresponding to Microarray Probes Showing Sexand Apo-specific (below) CNV.
Supplemental Figure 4. qRT-PCR Validation of 22 cDNAs Corresponding to Microarray Probes that Showed Differential CNV between
Sexual and Apomictic Boechera Genotypes.
Supplemental Table 1. List of 22 Boechera cDNAs and Their
Corresponding log2 ratios Selected across Two Apomictic and Two
Sexual Boechera Genotypes for qRT-PCR for the Validation of CNV
Data.
Supplemental Table 2. CNV Including No Copy Change, Depletions,
and Enrichment Derived from CGH of 730K cDNA Array Probes for 10
Apomixis and 10 Sexual Boechera Genotypes.
Supplemental Table 3. Top BLASTn Results for 31 Boechera cDNAs
Corresponding to Microarray Probes Identified as Being Best Discriminative between Sexual and Apomictic Genotypes.
Supplemental Table 4. Arabidopsis Gene Homologs, Characterized
by the “Pollen–Pistil Interactions (GO:0009875)” GO Term, Corresponding to Enriched Boechera Coding Regions.
ACKNOWLEDGMENTS
The authors acknowledge the technical support of Heidi Block, Leane
Boerner, and Vera Katzorke (Leibniz Institute of Plant Genetics and Crop
Plant Research), and thank Michael Windham (Duke University) and
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The Plant Cell
Marcus Koch (University of Heidelberg) for their help with species
identifications. We also thank M. M. Muraya and B. Knuepfer of the
Heterosis Group (Leibniz Institute of Plant Genetics and Crop Plant
Research) for their support; R. Bannen and J. Jeddeloh (Roche
NimbleGen) for their helpful ideas in the experimental design used
here; Thomas Thiel (Apomixis group—Leibniz Institute of Plant Genetics
and Crop Plant Research) for useful advice on experimental design and
analysis; and Ingo Schubert (Leibniz Institute of Plant Genetics and Crop
Plant Research) for constructive comments on the manuscript. Above all,
we thank the Deutsche Forschungsgemeinschaft (DFG SH 337/4-1 and
DFG SH 337/4-2) for the funding of this project (T.F.S. and O.M.A.), as
well as the Ministry of Culture of Saxony Anhalt for a grant (to M.S;
XP3624HP/0606T).
AUTHOR CONTRIBUTIONS
O.M.A. and J.M.C. generated the microarray data. M.S., O.M.A., and T.F.S.
analyzed the data. J.F. performed all genome size analyses. T.F.S. was
responsible for experimental design.
Received May 21, 2013; revised September 11, 2013; accepted October
15, 2013; published October 29, 2013.
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Copy Number Variation in Transcriptionally Active Regions of Sexual and Apomictic Boechera
Demonstrates Independently Derived Apomictic Lineages
Olawale M. Aliyu, Michael Seifert, José M. Corral, Joerg Fuchs and Timothy F. Sharbel
Plant Cell; originally published online October 29, 2013;
DOI 10.1105/tpc.113.113860
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