The Plant Cell, Vol. 26: 1448–1463, April 2014, www.plantcell.org ã 2014 American Society of Plant Biologists. All rights reserved.
Homoeologous Chromosome Sorting and Progression of
Meiotic Recombination in Brassica napus: Ploidy
Does Matter!
W
Laurie Grandont,a,b Nieves Cuñado,c Olivier Coriton,d Virgine Huteau,d Frédérique Eber,d Anne Marie Chèvre,d
Mathilde Grelon,a,b Liudmila Chelysheva,a,b and Eric Jenczewskia,b,1
a INRA,
UMR1318, Institut Jean-Pierre Bourgin, F-78000 Versailles, France
Institut Jean-Pierre Bourgin, F-78000 Versailles, France
c Departamento de Génetica, Facultad de Biología, Universidad Complutense, 28040 Madrid, Spain
d INRA, UMR 1349, Institut de Génétique, Environnement et Protection des Plantes, F-35653 Le Rheu Cedex, France
b AgroParisTech,
Meiotic recombination is the fundamental process that produces balanced gametes and generates diversity within
species. For successful meiosis, crossovers must form between homologous chromosomes. This condition is more
difficult to fulfill in allopolyploid species, which have more than two sets of related chromosomes (homoeologs). Here, we
investigated the formation, progression, and completion of several key hallmarks of meiosis in Brassica napus (AACC),
a young polyphyletic allotetraploid crop species with closely related homoeologous chromosomes. Altogether, our results
demonstrate a precocious and efficient sorting of homologous versus homoeologous chromosomes during early prophase
I in two representative B. napus accessions that otherwise show a genotypic difference in the progression of homologous
recombination. More strikingly, our detailed comparison of meiosis in near isogenic allohaploid and euploid plants showed
that the mechanism(s) promoting efficient chromosome sorting in euploids is adjusted to promote crossover formation
between homoeologs in allohaploids. This suggests that, in contrast to other polyploid species, chromosome sorting is
context dependent in B. napus.
INTRODUCTION
Meiosis is essential to the life cycle of all sexual eukaryotes; this
specialized type of cell division not only ensures fertility and
genome stability throughout sexual life cycles, but also generates diversity within species by creating new chromosome/
allele combinations. All these outcomes depend on the formation of meiotic crossovers (COs), one of the products of meiotic
double-strand break repair occurring during prophase I. At least
two classes of COs coexist in plants (Mézard et al., 2007). The
first class (class I COs) is dependent on the ZMM (for Zip, Msh,
Mer) group of proteins (Osman et al., 2011) and gives rise to
interfering COs, which are further apart from each other than
would be expected if they were independent. The second class
(class II COs) results in noninterfering COs and requires the
METHYL METHANESULFONATE and UV SENSITIVE81 and
METHYL METHANESULFONATE SENSITIVE proteins (Osman
et al., 2011). Due to their role as a physical link between chromosomes, at least one obligate CO is required between pairs of homologs (Jones 1984), which probably arise from the class I
pathway (Chelysheva et al., 2010). When this obligate CO is abolished, chromosomes segregate randomly, resulting in aneuploid
1 Address
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: Eric Jenczewski (eric.
[email protected]).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.114.122788
gametes, aneuploid progenies, and reduced fertility (MartinezPerez and Colaiácovo, 2009). The same negative outcome occurs
when COs are formed between multiple and/or nonhomologous
chromosomes, a situation that is more likely to occur in polyploid
species (reviewed in Grandont et al., 2013).
In polyploid species, every chromosome has more than one
potential partner, which can either be identical (when the polyploid results from the doubling of a single diploid genome, i.e.,
autopolyploidy) or slightly more divergent (when the polyploid has
an interspecific hybrid origin, i.e., allopolyploidy). In any case, the
different sets of chromosomes of a polyploid species must be
sorted out during meiosis to produce balanced gametes. As
a consequence, most allopolyploid species have inherited or
evolved recombination-modifying loci that suppress CO formation between the “homoeologous chromosomes” inherited from
the different parental species (Grandont et al., 2013, and references therein). The most well characterized of these loci is Pairing
homeologous1 (Ph1) from wheat (Triticum aestivum; Griffiths
et al., 2006), which was shown to affect CO formation both between homoeologs (Riley and Chapman, 1958; Greer et al.,
2012, and references therein) and homologs (Lukaszewski and
Kopecký, 2010).
In the past 10 years, Brassica napus has emerged as another
model for studying natural variation in CO frequency between either
homoeologs or homologs. B. napus (AACC, 2n=38) is a recent
allotetraploid species that originated from multiple interspecific
hybridization events between ancestors of Brassica oleracea (CC,
2n=18) and Brassica rapa (AA, 2n=20) (Allender and King, 2010, and
references therein). These two parental species share an ancestral
Meiotic Crossovers in Brassica napus
whole-genome triplication that occurred soon after the divergence
of the Brassica and Arabidopsis thaliana lineages (Lysak et al.,
2005). It has long been known that B. napus has a diploid-like
meiotic behavior, although a small number of homoeologous exchanges, usually referred to as translocations, have been detected
in several B. napus cultivars (Osborn et al., 2003; Udall et al., 2005;
Howell et al., 2008). However, their frequency remains very low,
especially when compared with the rate measured in newly created
synthetic B. napus allotetraploids (i.e., colchicine-doubled B. rapa 3
B. oleracea hybrids). The enhanced genomic stability of natural B.
napus could indicate that this species has inherited or evolved a locus
(or loci) suppressing/reducing CO formation between homoeologous
chromosomes. B. napus allohaploids also show significant variation
in meiotic behavior at metaphase I, with two main meiotic phenotypes identified across a wide range of varieties (Nicolas et al., 2009;
Cifuentes et al., 2010, and references therein). This variation relies on
polygenic control, with one major quantitative trait locus, named
Pairing regulator in Brassica napus (PrBn), being far more influential
than the others (Cifuentes et al., 2010, and references therein).
Our current understanding of CO variation in B. napus is mainly
based on cytological observations at metaphase I and genetic
surveys of intergenomic exchanges in the progenies of B. napus
allohaploids. As these approaches focused on the final products of
meiotic recombination (chiasmata and genetic exchanges), they did
not provide comprehensive information on the way(s) homologous
bivalents form in euploid B. napus or the causes for the observed
difference in CO rate between allohaploid plants. We do not even
know whether the same strategy for homologous bivalent formation is implemented in B. napus euploid accessions originating from
different polyploidization events (Cifuentes et al., 2010). The aim of
this study, therefore, was to specifically address these questions.
For this, we set up a thorough comparative analysis of meiosis in
euploid and allohaploid plants chosen to be representative of (1) the
different origins of B. napus and (2) the dichotomy of meiotic phenotypes observed among B. napus allohaploids. We investigated
the formation, progression, and completion of several key hallmarks
of meiosis, including sister chromatid cohesion, chromosome axes,
the synaptonemal complex, meiotic recombination, in order to determine the extent to which these factors contribute to the observed
differences or vary with the different origins of B. napus. Altogether,
our results demonstrate that homologous and homoeologous
chromosomes are sorted at an early stage of prophase I in two B.
napus genotypes, which otherwise show varying numbers of class I
COs. However, and contrary to other polyploid species, the
mechanisms regulating efficient chromosome sorting in euploids do
not suppress CO formation between homoeologs in the near isogenic allohaploids.
RESULTS
B. napus Euploids Show a Diploid-Like Meiotic Behavior
We first compared progression of male meiosis in Darmor-bzh
and Yudal euploid genotypes (with 2n=38 chromosomes, i.e.,
AACC), which are representative of the two main B. napus gene
pools (Harper et al., 2012). Staining chromosomes with 49,6diamidino-2-phenylindole (DAPI) showed that meiosis is very
regular in Darmor-bzh and Yudal euploids (Figure 1) and very
1449
similar to meiosis in Arabidopsis (Ross et al., 1996). Briefly, meiotic
chromosomes first condensed at leptotene (Figures 1A and 1K). At
zygotene, we observed several close alignments of chromosomes
(Figures 1B and 1L), which are diagnostic for the establishment of
the synaptonemal complex. At pachytene, all chromosomes were
closely aligned with one another (Figures 1C and 1M), indicating
that homologous chromosomes were fully synapsed (see below).
The synaptonemal complex then disassembled and chromosomes
condensed until discrete separate bivalents became visible at
diakinesis (Figures 1D and 1N). At metaphase I, we always observed 19 bivalents in the two genotypes with readily discernible
chiasmata, the cytological manifestation of meiotic COs (Figures 1E
and 1O). Based on chromosome shape (Jones 1984), we estimated
a mean cell chiasmata frequency (n = 20 pollen mother cells
[PMCs]) of 35 in both Darmor-bzh and Yudal (Table 1). The second
meiotic division then took place (Figures 1G to 1I and 1Q to 1S). It
resulted in four balanced sets of 19 chromatids (Figures 1J and 1T)
that invariably led to tetrad formation (n = 250 and 251 cells for
Darmor-bzh and Yudal euploids, respectively; Supplemental Figure
1). In both Darmor-bzh and Yudal, pollen grains were 100% viable
(Supplemental Figure 1), demonstrating that meiosis in B. napus
euploids leads to faithful chromosome segregation.
B. napus Allohaploids Show Impaired Meiosis but Still
Produce a Few Viable Pollen Grains
The early stages of meiosis in allohaploid plants (with 19 chromosomes, i.e., AC) appeared similar to those described in euploids.
The first noticeable difference occurred at pachytene (Figures 2A
and 2J). Both unaligned regions and stretches of juxtaposed
regions between pairs of nonhomologous chromosomes were
observed in all PMCs at this stage (hereafter referred to as
pachytene-like PMCs) in both Darmor-bzh (n = 52) and Yudal
(n = 59) allohaploids, suggesting partial synapsis (see below).
At diakinesis (Figures 2B and 2K), the chromosomes looked
diffuse, as if they were less condensed than in euploids, and they
were often entangled, especially in Yudal allohaploids. At metaphase I (Figures 2C and 2L), both univalents (i.e., chromosomes
that failed to form chiasmata) and bivalents were observed, with
variable numbers between genotypes. The number of chiasmata
was also significantly higher in Darmor-bzh (9.25 on average) than
in Yudal (5.70 on average) allohaploids (P = 0.0001; Table 1),
which is consistent with Cifuentes et al. (2010). In contrast to our
observations in euploids, these chiasmata were formed between
nonhomologous A and C chromosomes because allohaploids do
not contain homologous chromosomes.
Regardless of the number of chiasmata formed, in allohaploid
telophase I nuclei (Figures 2E and 2N), the chromosome composition was unequal, which, in most cases, led to unbalanced
tetrads (Figures 2H, 2Q, and 2R) or polyads (when laggard
chromosomes lead to the formation of additional nuclei) and,
thus, to unviable pollen grains (Supplemental Figure 2). Unexpectedly, meiosis in Darmor-bzh and Yudal allohaploids also
produced varying numbers of triads (Figure 2I) and dyads (Figure
2S); these were the minority in Darmor-bzh allohaploids (;5%),
but they represented half the products formed during meiosis in
Yudal allohaploids (Supplemental Figure 2). Immunolocalization of
a-tubulin (a component of the meiotic spindle) showed that the
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Figure 1. Meiosis in Euploid B. napus.
DAPI staining of pollen mother cells during meiosis of Darmor-bzh ([A] to [J]) and Yudal ([K] to [T]) euploids. Leptotene ([A] and [K]): Chromosomes condense
and become visible as unpaired threads. Zygotene ([B] and [L]): Arrows indicate several close juxtapositions of chromosomes that probably mark the initiation
of the synaptonemal complex. Pachytene ([C] and [M]): All chromosomes are closely aligned with one another, suggesting that the synaptonemal complex is
complete. Diakinesis ([D] and [N]): chromosomes are condensed and form discrete separate bivalents. Metaphase I ([E] and [O]): All bivalents aligned on the
metaphase plate. Anaphase I ([F] and [P]): Homologous chromosomes, each composed of two sister chromatids, move to the opposite poles. Chromosome
bridges are often observed at this stage but no chromosome fragmentation is observed at telophase I ([G] and [Q]). Metaphase II ([H] and [R]). Anaphase II ([I]
and [S]): Individual chromatids segregate to the spindle poles. Late anaphase II ([J] and [T]): Cells contain four sets of 19 chromatids. Bars = 5 mm.
dyads and triads resulted from the formation of parallel and/or
tripolar spindles during metaphase II (Supplemental Figure 3),
which regrouped the products of the first division. Thus, the resulting microspores contained the 19 chromosomes of the basic
B. napus chromosome set and probably generated the viable
pollen grains observed in the two allohaploid genotypes (see
pollen colored in red in Supplemental Figure 2).
Chromosome Axes Are Correctly Formed in Both Euploid
and Allohaploid B. napus
Next, we investigated sister chromatid cohesion and assembly
of meiosis-specific chromosome axes in B. napus, as these two
meiotic landmarks are essential for CO formation. For this, we
coimmunolocalized the meiosis-specific cohesin RECOMBINATION8
(SYN1/REC8) and the axis-associated protein ASYNAPTIC1 (ASY1),
which are both required for wild-type levels of COs in Arabidopsis
(Armstrong et al., 2002; Chelysheva et al., 2005).
REC8 and ASY1 staining was normal in both euploid and allohaploid B. napus (Supplemental Figures 4 and 5). The two proteins
were correctly loaded on chromatin at leptotene and progressively
formed a continuous signal as the chromosomes condensed and
synapsed. The two signals colocalized along the entire length of the
chromosome axes, whether they were synapsed or not in pachytene or pachytene-like cells. The signals persisted at diakinesis and
metaphase I in both allohaploid and euploid B. napus.
These results suggest that sister chromatin cohesion and
chromosome axes are correctly established and maintained in
Meiotic Crossovers in Brassica napus
1451
Table 1. Progression of Meiotic Recombination in B. napus Euploids (AACC; 2n=38) and Allohaploids (AC; n = 19)
Genotypes
Darmor-bzh
euploids
Yudal
euploids
PMCs at Pachytene
PMCs at Diplotene
PMCs at Diakinesis
No. of
PMCs
Avg. No. of
HEI10 Focia
No. of
PMCs
No. of
PMCs
13
27.46 (16–36)
7
23.86 (20–28)
37
28.05 (18–38)
11
29.09 (25–36)
n.s.
24.20 (14–40)
–
n.s.
–
16.41 (7–28)
–
–
Darmor-bzh
25
allohaploids
Yudal
71
allohaploids
Avg. No. of
HEI10 Foci
PMCs at Metaphase I
Avg. No. of
MLH1 Foci
Avg. No. of HEI10
+ MLH1 Foci
No. of
PMCs
Avg. No. of
Bivalents
Avg. No. of
Chiasmata
33
–
34.82 (25–45)
20
19
35.45
29
–
31.00 (21–38)
20
19
35.05
207
18.34 (3–39)
**
6.24 (1–10)b
19
6.67 (5–8)
8.92
134
10.27 (4–25)
2.47 (1–5)b
20
5.00 (3–8)
5.70
**
**
**
**
Values in parentheses indicate the range of variation. n.s., not significantly different (Student’s t test). **, Significantly different (Student’s t test).
a
These values were obtained for PMCs showing only distinct HEI10 foci.
b
These values were obtained for 17 and 30 PMCs for Darmor-bzh and Yudal allohaploids, respectively.
the two genotypes. As no difference was observed between
Darmor-bzh and Yudal, the difference in meiotic behaviors observed at metaphase I in allohaploids is not due to an obvious defect in chromosome axis formation. Chromosome shape (based on
DAPI staining) and ASY1 staining were also instrumental for
discriminating between the different prophase I-like stages in
allohaploids (Supplemental Figure 6). The chromosomes in early
zygotene PMCs had a compact appearance and were strongly
labeled with the anti-ASY1 antibody, whereas in pachytene-like
PMCs, chromosomes looked less compact and a continuous ASY1
signal was observed. The chromatin in late diplotene PMCs was
very diffuse and the ASY1 signal began to disappear from the
chromosome axes. These criteria were used in the rest of our study.
The Extent and Nature of Synapsis Is Different in B. napus
Euploids and Allohaploids
We then used two complementary approaches to gain a better
understanding of synaptonemal complex formation in B. napus
euploids (Figure 3). First, to monitor the progression of synapsis, we
immunolocalized ZIPPER1 (ZYP1), a component of the transverse
filament of the synaptonemal complex in A. thaliana (Higgins et al.,
2005). Second, we analyzed surface-spread prophase I nuclei with
electron microscopy to examine whether synaptic multivalents are
formed prior to or at pachytene.
The ZYP1 signal overlapped with the ASY1 signal along the
entire length of the chromosomes in both Darmor-bzh and Yudal
euploids (Figures 3A and 3B). This indicates that full synapsis does
occur at pachytene in the two genotypes (n = 15 and n = 18 PMCs,
respectively), even if the signal in Yudal was never as continuous as
in Darmor-bzh. Electron microscopy observations confirmed that
synapsis is complete in the two genotypes with no apparent difference between them (59 and 43 nuclei from late zygotene to
pachytene were reconstructed for Darmor-bzh and Yudal euploids,
respectively). In the two genotypes, 19 synapsed bivalents, which
most likely correspond to homologous pairs, were observed only in
;50% of nuclei (Figures 3C and 3D, Table 2). In the other ;50% of
nuclei, one (40% of nuclei) or two (;10% of nuclei) synaptic
quadrivalents, formed by the association of four chromosomes
joined at different points, were observed, with no difference
between the two genotypes (Figures 3E to 3G, Table 2;
Supplemental Figure 7). The majority of these synaptic quadrivalents had only one synaptic partner switch (Figures 3E and
3G), and only 15% had two synaptic partner switches (Figure
3F). Most synaptic partner switches were preferentially located
near the chromosome ends (subterminal or distal position),
while a few occurred in more interstitial regions. We never
observed a synaptic partner switch within the central part of
the chromosomes. When synaptonemal complexes were
measured in nuclei showing no or little distortion using electron
microscopy (i.e., no interlock, no synaptonemal complex
fragmentation, and no foldback loop), no significant difference
in synaptonemal complex length between Darmor-bzh and
Yudal euploids was found (532 6 20 versus 503 6 25 mm,
respectively; Student’s t test, P = 0.389).
Synaptonemal complex formation in Darmor-bzh and Yudal
allohaploids was examined using the same two approaches as for
the euploids (Supplemental Figure 8). This confirmed that synapsis
is never complete in allohaploids. Although several continuous
stretches of ZYP1 signal were observed in both Darmor-bzh and
Yudal allohaploids, most of these tracts were shorter than the
chromosome length and were systematically accompanied by
unsynapsed chromosome axes labeled by ASY1, but not ZYP1 (n =
16 and n = 15 PMCs, respectively). Nevertheless, electron microscopy did reveal the presence of one or two completely synapsed bivalents (Supplemental Figure 8) in ;50% of prophase I
nuclei, amid partially synapsed or completely unsynapsed chromosomes. These complete synaptic bivalents appeared to be as
frequent in Darmor-bzh as in Yudal allohaploids (n = 46 and n = 35
PMCs, respectively).
The absence of complete synapsis made comparison between Darmor-bzh and Yudal allohaploids difficult; cell-to-cell
differences could reflect either differences in the extent of synaptonemal complex completion or differences in the stages that
were observed. With this limitation in mind, no obvious difference in synaptic behavior was found between Darmor-bzh and
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The Plant Cell
Figure 2. Meiosis in Allohaploid B. napus.
DAPI staining of pollen mother cells during meiosis of Darmor-bzh ([A] to [I]) and Yudal ([J] to [S]) allohaploids. Pachytene ([A] and [J]): The presence of
chromosomes that are not closely juxtaposed with one another (arrows) suggests that the synaptonemal complex is incomplete. Diakinesis ([B] and
[K]). Metaphase I ([C] and [L]) with variable numbers of bivalents and univalents. Anaphase I ([D] and [M]): Nonhomologous chromosomes, each
composed of two sister chromatids, are separated. At this stage chromosome bridges are often observed as in euploids. Telophase I ([E] and [N]): Two
groups of chromosomes are observed, indicating that univalents moved to one or the other pole of the cell. Metaphase II ([F] and [O]). Anaphase II ([G]
and [P]): Individual chromatids segregated to the spindle poles resulting in the formation of different kinds of meiotic products, including unbalanced
tetrads ([H], [Q], and [R]), triads (I), as well as dyads (S). Bars = 5 mm.
Yudal allohaploids with either immunolocalization or electron
microscopy.
The Dynamics of Class I CO Formation Is Different
between the Two Genotypes
We monitored the progression of meiotic recombination by
immunostaining HUMAN ENHANCER OF INVASION10 (HEI10),
a key protein that is unique because it allows the progressive
transition from early recombination intermediates into final class I
COs to be scrutinized (Chelysheva et al., 2012).
HEI10 showed the same dynamic localization pattern during
prophase I in B. napus euploid plants as in A. thaliana (Chelysheva
et al., 2012) and rice (Oryza sativa; Wang et al., 2012). At zygotene,
multiple HEI10 foci were present along chromosome axes (Figure
4A; Supplemental Figure 9A). During pachytene, the number of
HEI10 foci dropped dramatically, changing from a zygotene-like
pattern (Figure 4B; Supplemental Figure 9B) until only a few distinct foci persisted on chromatin (Figure 4C; Supplemental Figure
9C). At diplotene (Figure 4D; Supplemental Figure 9D), distinct
HEI10 foci were still present on the chromosomes where they
systematically colocalized with MUT L HOMOLOG1 (MLH1) foci,
a marker of class I COs (Chelysheva et al., 2010) at diakinesis
(Figure 4E; Supplemental Figure 9E). Thus, HEI10 staining in
B. napus euploids is progressively restricted to the sites where
class I COs mature.
Meiotic Crossovers in Brassica napus
1453
Figure 3. Synaptonemal Complex Formation in B. napus Euploids.
(A) and (B) Chromosomes at pachytene were labeled with ASY1 (red) and ZYP1 (green) antibodies in Darmor-bzh (A) and Yudal (B) PMCs, respectively.
An overlay of the ASY1 and ZYP1 signals is shown (merge). Bars = 10 mm.
(C) and (D) Electron micrographs of silver stained late-zygotene nucleus in Darmor-bzh (C) and pachytene nucleus in Yudal (D) showing only synaptic
bivalents. Bars = 5 mm.
(E) to (G) High-magnification electron micrographs of quadrivalents in pachytene nuclei of Darmor-bzh ([E] and [F]) and Yudal (G) euploids with one ([E] and [G])
and two (F) synaptic partner switches and their corresponding schematic drawings. Arrows indicate synaptic partner switches. IL, interlocking. Bars = 2 mm.
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The Plant Cell
Table 2. Synaptic Associations in B. napus Euploids (AACC; 2n=38) at
Late Zygotene-Pachytene
Total No.
of PMCs
Number of PMCs with
Genotypes
19 II
17 II + 1 IV
15 II + 2 IV
Darmor-bzh
euploids
Yudal euploids
28 (47.5%)
24 (40.7%)
7 (11.8%) 59
19 (44.2%)
20 (46.5%)
4 (9.3%)
43
II, bivalents; IV, quadrivalents. Values in parentheses indicate the
proportion of cells in the different classes.
This HEI10 dynamic pattern was observed in both Darmor-bzh
and Yudal euploids. However, two important differences were
found. First, the pronounced decline in HEI10 foci was even
stronger in Yudal than in Darmor-bzh (Figure 5). In Darmor-bzh
euploids, 82% (60 out of 73) and 46% (6 out of 13) PMCs still
showed numerous HEI10 foci along chromosome axes at
pachytene and diplotene, respectively (Figure 5B; Supplemental
Figure 10). By contrast, in Yudal euploids, the vast majority of
PMCs (95% at pachytene, 37 out of 39; 100% at diplotene)
showed only distinct HEI10 foci (Figure 5G). No significant difference was found when the number of HEI10 foci was compared
in Darmor-bzh and Yudal pachytene PMCs that showed only
distinct HEI10 foci (Student’s t test; P = 0.7); only a moderate
difference was found at diplotene (P = 0.007).
Second, the mean number of foci where HEI10 and MLH1
colocalized at diakinesis was higher in Darmor-bzh (;35 foci;
n = 33 PMCs) than in Yudal euploids (;31 foci; n = 29 PMCs).
This small difference was statistically significant (Student’s
t test; P = 0.0014), suggesting that Darmor-bzh and Yudal differ
slightly in their propensity to form interfering COs. Indeed,
Darmor-bzh euploids also showed more bivalents with two,
three, or more MLH1 foci than Yudal (Fisher’s exact test, P =
1.65 1025; Supplemental Table 1).
We used the same approaches to compare the progression of
meiotic recombination in Darmor-bzh and Yudal allohaploids
(Figure 6; Supplemental Figure 11) and observed essentially the
same HEI10 patterns as in the corresponding euploids. Of note,
we observed the same difference in HEI10 dynamics at pachytene
(Figure 5). In Yudal allohaploids, distinct HEI10 foci typical of the
late HEI10 pattern were seen in 90% (71 out of 79) of pachytenelike PMCs, whereas these were observed in only 25% (25 out of
98) of PMCs in Darmor-bzh allohaploids. The largest HEI10 foci
observed in B. napus allohaploids were never found on unsynapsed regions even at the end of pachytene (Figure 6C;
Supplemental Figure 11C). This suggests that the formation of
distinct HEI10 foci occurs only on synapsed regions.
Despite these similarities, we found three differences between
euploids and allohaploids. First, in allohaploids but not euploids,
the mean number of HEI10 foci in pachytene-like PMCs was
significantly higher in Darmor-bzh than in Yudal (24.2 versus 16.4;
P < 0.001). This was mainly due to a significant decrease in the
number of HEI10 foci in Yudal allohaploids compared with Yudal
euploids (Table 1; P < 0.001), whereas the estimates were the
same in Darmor-bzh allohaploids and euploids (Table 1; P = 0.19).
Second, almost twice as many MLH1 foci were seen in
Darmor-bzh than Yudal allohaploids (on average, 18 versus 10
foci, respectively; Student’s t test, P < 0.001; Table 1, Figure 6E;
Supplemental Figure 11E), while the variation was much smaller
between euploids. Indeed, the distribution of MLH1 foci per cell
fit a Poisson distribution in the two allohaploids but not in the
euploids, in which an excess of counts near the mode and large
deficiencies at the extremes of the distribution were found
(Supplemental Figure 12). This strongly suggests that the distribution of MLH1 foci was random between PMCs in allohaploids, while it was constrained in euploids; this also explains
why data counts were more widely dispersed in allohaploids
than in euploids (Table 1).
Finally, coimmunolocalization of MLH1 with HEI10 revealed
that the two proteins only rarely colocalized at diakinesis in allohaploids (Figure 6F), but systematically did so in the corresponding euploids (Figure 4E; Supplemental Figure 9E). Distinct
and separate MLH1 and HEI10 foci were repeatedly observed at
diakinesis in three independent experiments, in both Darmor-bzh
and Yudal allohaploids. While the identity of the recombination
intermediates marked by the distinct and separate MLH1 and
HEI10 foci remain unknown, the two proteins colocalized in more
foci in Darmor-bzh than in Yudal allohaploids (6.2 versus 2.5; P <
0.001). Thus, a significant difference was found between the two
allohaploids regardless of which criteria were used to assess
class I CO formation (number of HEI10 foci, number of MLH1 foci,
number of MLH1+HEI10 foci; Table 1).
Finding a Partner to Recombine with: Chromosome- and
Genotype-Specific Effects
Finally, we characterized recombination between individual chromosomes during meiosis in allohaploid B. napus. For this, BAC
fluorescence in situ hybridization (FISH) experiments were performed using some of the BAC clones identified by Xiong et al.
(2010), which specifically and simultaneously label pairs of homoeologous chromosomes/regions. We first focused on the A1-C1
pair of homoeologs (Figure 7), which are collinear along their entire
length (Parkin et al., 2005). A1 and C1 were frequently held together
by COs in Darmor-bzh and Yudal allohaploids (Figures 7A and 7B,
Table 3), with no significant difference between the two genotypes
(P = 0.25). However, in Darmor-bzh allohaploids, chiasmata bound
the two arms (thus, at least two COs) of a slightly higher proportion
of bivalents than in Yudal allohaploids (72.3% versus 60.4%). In the
few PMCs in which A1 and C1 did not recombine together at
metaphase I in Darmor-bzh (10 PMCs out of 129; 7.8%) or Yudal
(nine PMCs out of 57; 15.8%) allohaploids, the chromosomes
formed either a bivalent with another chromosome (Figure 7C) or
remained univalent (Figure 7D).
We then extended our BAC FISH survey to three other pairs of
chromosomes that share homoeology only along one of their arms,
i.e., the long arms of A3/C3, the long arms of A7/C6, and the long
arm of A10/short arm of C9 (Parkin et al., 2003, 2005; Xiong et al.,
2010). Every pair showed a distinct and specific pattern at metaphase I (Supplemental Figure 13): A10 and C9 recombined together
much more frequently in Darmor-bzh than in Yudal allohaploids
(Table 3; P = 0.0095); A7 and C6 recombined together in the same
proportion of PMCs in Darmor-bzh and Yudal allohaploids (Table 3;
Meiotic Crossovers in Brassica napus
1455
Figure 4. Progression of Class I CO Formation in Darmor-bzh Euploids.
(A) to (D) Immunolocalization of ASY1 and HEI10 at zygotene (A), pachytene ([B] and [C]), and diplotene (D). Chromosomes were labeled with DAPI
(white, false color), and HEI10 (green) and ASY1 (red) antibodies. An overlay of the three signals is shown (merge).
(E) Immunolocalization of MLH1 and HEI10 at diakinesis. Chromosomes were labeled with DAPI (white, false color), and HEI10 (red) and MLH1 (green)
antibodies. The overlay of three signals is shown (merge).
Bars = 5 mm.
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The Plant Cell
Figure 5. The Dynamics of Class I CO Formation Is Different between Darmor-bzh and Yudal Euploids.
The proportion of PMCs showing either a high number of HEI10 foci along the entire length of chromosome axes (in green) or a small number of distinct
HEI10 foci on chromosome axes (in orange) is shown for Darmor-bzh euploid ([A] to [D]) and Yudal euploid ([E] to [H]). Bars = 5µm.
Figure 6. Progression of Class I CO Formation in Darmor-bzh Allohaploids.
(A) to (C) Immunolocalization of ASY1 and HEI10 at zygotene (A) and pachytene ([B] and [C]). Chromosomes were labeled with DAPI (white, false color),
and HEI10 (green) and ASY1 (red) antibodies. An overlay of the three signals is shown (merge).
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DISCUSSION
Early Homoeologous Chromosome Sorting in
B. napus Euploids
Figure 7. BAC FISH Survey of CO Formation for a Given Pair of
Homoeologous Chromosomes (A1-C1) in B. napus Allohaploids.
Chromosomes were stained with DAPI (white, false color), and FISH was
performed using two BACs that specifically and simultaneously identify
A1 and C1 chromosomes (red and green). Metaphase I PMCs showing
either only one bivalent labeled, indicating that A1 and C1 recombined
together ([A] and [B]) or showing two labeled entities indicating that A1
and C1 did not recombine together ([C] and [D]). In (C), two bivalents are
labeled, indicating that A1 and C1 recombined with other chromosomes
(arrows). In (D), two univalents are labeled, indicating that A1 and C1 did
not recombine. Bars = 5 µm.
P = 0.7512); A3 and C3 recombined together a little more often in
Yudal than in Darmor-bzh allohaploids (Table 3; P = 0.1915). We
were also able to distinguish a series of other bivalents, for which
only one operating chromosome was identified by the probes we
used (e.g., A3-Cx, C9-Ax.). These additional bivalents more
frequently involved A3 and/or C3 in Darmor-bzh allohaploids
and A10 and/or C9 in Yudal allohaploids. This suggests that
chromosome- and genotype-specific factors can change the
odds of forming a CO between a given pair of homoeologous
regions irrespective of the overall difference in CO rate between
Darmor-bzh and Yudal allohaploids.
Cytological diploidization of allopolyploid species can be
achieved in different ways, depending on the stage when homologous and homoeologous chromosomes are successfully
split apart (Jenkins and Rees, 1991). In this study, we showed
that homoeologous chromosomes are sorted early on during
prophase I in B. napus, synapsis being mostly restricted to
homologs by late zygotene-pachytene in both Darmor-bzh and
Yudal euploids. We observed no more than one or two synaptic multivalents in half of the PMCs observed at these stages
(Table 2), the origin of which can be interpreted in two different
ways.
The most straightforward hypothesis is that the synaptic multivalents result from synaptonemal complex formation between
homoeologous regions. In that case, B. napus would be like wheat,
Aegilops, and Festuca polyploids, in which varying numbers of
homologous and homoeologous chromosomes are partially synapsed during prophase I (Hobolth, 1981; Thomas and Thomas,
1993; Cuñado et al., 1996). Given that, in plants, the synaptonemal
complex does not form when strand invasion is impaired (Osman
et al., 2011), the presence of homoeologous synaptic associations
most likely indicates that strand exchanges are initiated between
homoeologs in B. napus euploids. This is consistent with the
demonstrated propensity for homoeologous chromosomes to recombine with one another in allohaploids (Figures 2 and 5). However, given the limited extent to which synaptic multivalents
persisted to pachytene in B. napus euploids, it appears that most
interhomoeolog recombination intermediates abort early and are
probably redirected into intersister or noncrossover pathways
(Hunter and Kleckner, 2001). Evidence for very short nonreciprocal
exchanges between homoeologous sequences, which possibly
originated from meiotic noncrossovers, was recently obtained
in cotton (Gossypium hirsutum), another bivalent-forming allopolyploid species (Salmon et al., 2010; Flagel et al., 2012). Further
studies are needed to demonstrate that similar events occur in B.
napus.
As an alternative, the synaptic multivalents observed at pachytene may result from the presence of translocated A/C chromosome(s), in which one region has been replaced by its homoeolog
that is thus duplicated within the genome. In that case, which was
first advocated by Osborn et al. (2003), the synaptonemal complex
could be formed only between homologous segments, albeit
carried by different chromosomes; this would mean that B. napus
Figure 6. (continued).
(D) Immunolocalization of HEI10 at diplotene. Chromosomes were labeled with DAPI (white, false color) and HEI10 (green) antibodies. An overlay of the
two signals is shown (merge).
(E) Immunolocalization of MLH1 at diakinesis. Chromosomes were labeled with DAPI (white, false color) and MLH1 (green) antibodies. The overlay of
two signals is shown (merge).
(F) Immunolocalization of MLH1 and HEI10 at diakinesis. Chromosomes were labeled with DAPI (white, false color), and HEI10 (red) and MLH1 (green)
antibodies. The overlay of three signals is shown (merge). Arrows indicate stand-alone MLH1 foci, arrowheads indicate HEI10 foci, and asterisks show
the colocalization of MLH1 and HEI10 foci.
Bars = 5 mm.
Meiotic Crossovers in Brassica napus
1459
Table 3. Homoeologous Bivalent Formation in B. napus Allohaploids (AC; n = 19) at Metaphase I
Frequencies of PMCs with a Bivalent between
Genotypes
A1-C1
A10-C9
A3-C3
A7-C6
Darmor-bzh allohaploids
Yudal allohaploids
92.8% (119/129)
84.2% (48/57)
80% (16/20)
35% (7/20)
53% (18/34)
71.4% (20/28)
40% (8/20)
50% (10/20)
Values in parentheses indicate the number of PMCs over the total number of PMCs observed.
homoeologous regions are sorted at an even earlier stage, or even
more efficiently than envisaged above. This is exactly what happens in oat (Avena sativa), Avena marrocana, and Allium montanum, in which the synaptonemal complexes appear to be
confined to homologous bivalents from zygotene onwards (Loidl,
1988; Jones et al., 1989). Although to date no A/C translocations
have been described in Darmor-bzh and Yudal, their existence
would help explain some of our BAC FISH observations; because
the chance for CO formation is higher between duplicated homologous segments than between homoeologous regions, translocated A/C chromosome(s) (e.g., presumably A3/C3 in Yudal) are
expected to recombine much more systematically than nonrecombinant chromosomes (Table 3). Thus, more work is needed
to confirm the presence of translocated A/C chromosome(s) in
Darmor-bzh and Yudal and their possible consequence on the
synaptonemal complex.
Regardless of the origin of the synaptic multivalents, our results
suggest that, in B. napus euploids, homoeologous chromosomes
are sorted prior to, or during, formation of stable strand exchanges,
which are thought to occur during zygotene concomitant with
synaptonemal complex formation (Hunter and Kleckner, 2001; Tiang
et al., 2012). The remaining multivalents are then eliminated before
diakinesis, as if a second layer of control suppresses CO formation
within the “illegitimately” synapsed regions. This latter adjustment
probably remains an error-prone process and a few COs can be
expected to occasionally form between homoeologs in euploids, in
the regions where synapsis first took place. Our results suggest that
these are fairly rare events (Table 1), although we have no evidence
that COs never occur between homoeologs in B. napus euploids.
Chromosome Sorting Is Context Dependent in B. napus
In wheat and oat, the sorting of homoeologous chromosomes in
euploids is paralleled by an almost complete suppression of CO
formation between homoeologs in the corresponding allohaploids
(Riley and Chapman, 1958; Gauthier and McGinnis, 1968). In these
plants, even if chromosomes have no choice but to recombine with
their homoeologs, they are prevented from doing so by loci responsible for the cytological diploidization of the euploid forms
(Griffiths et al., 2006; Greer et al., 2012, and references therein). Our
results show that the situation is strikingly different in B. napus; the
homoeologous synaptic and chiasmatic associations that are suppressed in euploids become dominant in allohaploids. This indicates
that the mechanism(s) responsible for the early sorting of homoeologous chromosomes in euploid B. napus do(es) not suppress
CO formation between homoeologous chromosomes in allohaploids. This does not mean, however, that there is no sorting
process operating in B. napus allohaploids. On the contrary, COs
are preferentially formed between homoeologs in these plants and
more occasionally between the duplicated blocks inherited from the
Brassica ancestral whole-genome triplication (i.e., paleologs)
(Nicolas et al., 2009, and references therein). Thus, it is as if a flexible
but efficient sorting mechanism is adjusted to promote CO formation between the closest available matches (homologs in euploids
and homoeologs in allohaploids), disregarding the other potential
partners (homoeologs in euploids; paleologs in allohaploids).
Our detailed comparison of meiosis in euploid versus allohaploid
B. napus thus suggests that the threshold for committing a pair of
chromosomes to form a CO is not fixed once and for all in B. napus,
but depends on the operating chromosomes. As mentioned above,
this requires that only a fraction of recombination intermediates is
set to become mature, a decision that cannot be made only locally
(e.g., based on the stability of DNA heteroduplexes) because otherwise the intermediates that are committed to form COs in allohaploids could hardly be aborted in euploids. Instead, homology
appears to be processed along the entire chromosome length. This
destabilizes the weakest or less common nascent recombination
intermediates formed between the most divergent chromosomes
and promotes the strongest or more numerous intermediates that
were formed, from the outset, between the least divergent chromosomes. As the outcome of this competition depends on the
operating chromosomes, the most likely winners are expected to
change with context.
The mechanism(s) for this “context-dependent chromosome
sorting” is (are) unknown but appear(s) to be as efficient or work
with almost the same level of stringency in Darmor-bzh and
Yudal (see below). Indeed, no clear difference was observed
between the two genotypes, which showed almost the same
proportion of pachytene PMCs with 19 synapsed bivalents and
the same frequency of synaptic multivalents (Table 2). Why,
then, are there different numbers of meiotic COs between homoeologous chromosomes in allohaploid plants (Table 1)?
Variation in Class I CO Numbers between
B. napus Varieties
In this study, we first demonstrated that the spatial-temporal
localization of HEI10, a ZMM protein essential for CO formation in plants, is the same during prophase I in B. napus euploids as in A. thaliana and rice (Chelysheva et al., 2012; Wang
et al., 2012). Thus, as with these two species, the dynamic
relocation of HEI10 in B. napus probably reflects the progressive channeling of recombination intermediates into the
ZMM CO pathway (see Chelysheva et al., 2012 for details). We
then showed that this progression varies from one genotype
to another. The transition from early to late HEI10 staining
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The Plant Cell
appeared sharper in Yudal than in Darmor-bzh (Figure 5). Finally,
immunolocalization of MLH1 demonstrated that Darmor-bzh and
Yudal form different numbers of class I COs (Table 1). Although it
remains to be established whether the different patterns and/or
chronology of HEI10 relocation contribute to this variation in class I
COs, our results suggest that there are more COs in Darmor-bzh
than in Yudal, all things being equal. Does it follow that allohaploids
with higher CO frequencies have originated from allotetraploid
accessions also showing higher numbers of homologous COs?
Interestingly, B. napus allohaploids displayed the same
zygotene-to-diplotene patterns of HEI10 staining as the corresponding euploids (Figure 6; Supplemental Figure 11). This suggests that the progression of early meiotic recombination is
essentially the same whether recombination intermediates are
formed between homologs or homoeologs (provided, however,
that there is no homolog to compete with them; see above).
However, (late) distinct HEI10 foci were only found on synapsed
regions in allohaploids (Figure 6C; Supplemental Figure 11C),
suggesting that these are probably dependent on synaptonemal
complex formation or conversely required to nucleate synaptonemal complex (see Reynolds et al. [2013] for comparable results
in mice). Even more strikingly, we observed that the number of
MLH1 foci per cell fit a Poisson distribution in the two allohaploids,
as if they were distributed at random between PMCs. As first
proposed by Jones (1967), this distribution may actually reflect
a breakdown of the process(es) regulating the distribution of MLH1
foci at the bivalent level. In line with this hypothesis, we observed
that only a small fraction of HEI10 and MLH1 foci colocalized at
diakinesis in B. napus allohaploids (Figure 6F), while this was
systematic in euploids (Figure 4E; Supplemental Figure 9E). The
nature of these separate HEI10 and MLH1 foci is unknown, but it is
probably no coincidence that they were found in plants showing
partial synapsis (Qiao et al., 2012, and references therein). As recently proposed for the tomato (Solanum lycopersicum) asynaptic
mutant as1 (Qiao et al., 2012) or haploid Arabidopsis (Cifuentes
et al., 2013), the “stand-alone” MLH1 foci could in fact mark the
locations where COs eventually failed or occurred between sister
chromatids. As there is bias against forming COs between sister
chromatids (at least in haploid Arabidopsis; Cifuentes et al., 2013),
it is possible that a fraction of late recombination intermediates are
still in the process of being resolved at diakinesis in B. napus
allohaploids, resulting in some HEI10 foci persisting longer than
usual on chromosomes without producing the conditions required
for MLH1 loading. Finally, separate HEI10 and MLH1 foci may
reflect an aberrant turnover of meiotic proteins in allohaploid
plants, with MLH1 loading and off-loading irrespective of HEI10.
Regardless of the causes and consequences of separate HEI10
and MLH1 foci, there were significantly more HEI10 foci, more
MLH1 foci, and more “MLH1+HEI10” foci in Darmor-bzh allohaploids than Yudal allohaploids (Table 1; Supplemental Figure 12).
Variation in class I CO rates may thus explain a significant proportion of the variation in total CO rate observed between Darmorbzh and Yudal allohaploids. It is of note, however, that the mean
number of distinct HEI10 foci is significantly lower at pachytene in
Yudal allohaploids compared with Yudal euploids (16.4 versus 28.0
foci; P < 0.001; Table 3), while it is almost the same in Darmor-bzh
allohaploids and euploids (24.20 versus 27.46 foci; P = 0.19;
Table 3). This suggests that fewer double-strand breaks are
committed to form COs between homoeologous chromosomes in
Yudal allohaploids than in euploids (or in Darmor-bzh). Thus, our
results point toward a possible difference in the sensitivity to
chromosome divergence between the two genotypes.
In fact, it may be pointless to consider suppression of COs between homoeologs and variation in CO number between homologs
as separate processes in allopolyploid species. There is some indication, for instance, that Ph1, the main locus responsible for the
cytological diploidization of wheat (Riley and Chapman, 1958;
Griffiths et al., 2006; Greer et al., 2012, and references therein), affects recombination between dissimilar homologs (Lukaszewski and
Kopecký, 2010, and references therein), suggesting that this locus
regulates some basic mechanism of chromosome recognition
(Greer et al., 2012). Whether the same holds true for PrBn and B.
napus is an avenue worth exploring.
METHODS
Plant Material and Growth Conditions
The production of allohaploid plants from Brassica napus cv Darmor-bzh and
Yudal was described by Cifuentes et al. (2010).The plants were cultivated in
a greenhouse or growth chamber under a 16-h-light/8-h-night photoperiod, at
22°C day and 18°C night, with 65% humidity.
Cytology
Male meiotic spreads for DAPI staining were prepared as described by
Chelysheva et al. (2013) from buds fixed in Carnoy’s fixative (absolute
ethanol:acetic acid, 3:1, v/v).
Final male meiotic products were observed by toluidine blue staining
as described by Azumi et al. (2002), and mature pollen grain viability was
estimated as described by Alexander (1969). Images were taken with
a Leica Diaplan bright-field microscope.
Immunolocalization of a-Tubulin
Anthers dissected from fixed buds at the appropriate meiotic stage were
stained according to a protocol adapted from Mercier et al. (2001). Buds were
incubated for 5 min in citrate buffer, pH 6.0, and rinsed in water. After a first
digestion of 1 h in digestion mix (0.3% [w/v] cellulase RS, 0.3% [w/v] pectolyase
Y23, and 0.3% [w/v] cytohelicase in citrate buffer), one anther was placed on
a polysin slide, dissected in water, squashed, and fixed in nitrogen. The released
cells were immobilized with a thin layer of 1% gelatin (w/v), 1% agarose (w/v),
and 7% glucose, incubated for 5 min in citrate buffer, pH 6.0, and digested for
a further 1 h in digestion mix at 37°C. After three rinses in 0.1% PBS-T (10 mM
sodium phosphate, pH 7.0, 143 mM NaCl, and 0.1% Triton X100), cells were
incubated for 1 h and 30 min in 1% PBS-T (10 mM sodium phosphate, pH 7.0,
143 mM NaCl, and 1% Triton X100) at room temperature and rinsed two times
with 0.1% PBS-T. Cells were incubated overnight at 4°C with an anti-a-tubulin
mouse monoclonal antibody (Sigma-Aldrich) diluted 1/325 in PBS-T-BSA (1%
BSA in 0.1% PBS-T). After three rinses with 0.1% PBS-T, cells were incubated
for 2 h and 30 min in fluorescein isothiocyanate–labeled secondary antibody
(labeled goat anti-mouse IgG Alexa fluor 488) diluted 1/100 in PBS-T-BSA at
37°C. After three rinses in 0.1% PBS-T, the cells were mounted in Vectashield
antifade medium (Vector Laboratories) with 2 mg/mL DAPI.
Immunolocalization of Meiotic Proteins
Coimmunolocalization of ASY1/REC8, HEI10/ASY1, and MLH1/HEI10 was
performed on meiotic spreads prepared as described previously for DAPI
staining. Immunolabeling was performed as described by Chelysheva et al.
Meiotic Crossovers in Brassica napus
(2013). The coimmunolabelingof ASY1/ZYP1 was performed as described by
Chelysheva et al. (2007) from fresh buds. The anti-ASY1 polyclonal antibody
has been described elsewhere (Armstrong et al., 2002). It was used at a dilution of 1:250. The anti-REC8 polyclonal antibody (Cai et al., 2003) was used
at a dilution of 1:250. The anti-HEI10 polyclonal antibody (Chelysheva et al.,
2012) was used at a dilution of 1:200. The anti-MLH1 antibody (Chelysheva
et al., 2012) was used at a dilution of 1:20 (purified serum). The anti-ZYP1
polyclonal antibody (Higgins et al., 2004) was used at a dilution of 1:500.
BAC FISH Experiment
BAC FISH experiments on meiotic spreads (see above) were performed as
described by Lysak et al. (2006) with the following modifications.
Probe Labeling
To label A1/C1, A3/C3, A7/C6, and A10/C9 homoeologous chromosomes,
five BACs from Brassica rapa (KbrB036M17 and KbrB052L10 for A1/C1;
KbrH117M18 for A3/C3; KbrB21P15 for A7/C6; and KbrH80A08 for A10/
C9) that were shown to be specific for A1 in B. rapa (Kim et al., 2009) and
expected to hybridize to homoeologous regions of the C genome of B.
napus (Xiong and Pires, 2011) were used. The five BACs were labeled using
Biotin-Nick Translation Mix (Roche) and DIG Nick Translation Mix (Roche) as
described by Lysak et al. (2006) or random priming with Alexa 488-5-dUTP
and biotin-14-dUTP (Invitrogen, Life Technologies) (Suay et al., 2013). The
labeled BACs were then precipitated and the dry pellet dissolved in 10 mL
HB50 (50% deionized formamide, 23 SSC [0.30 mM NaCl and 0.030 M
Na3-citrate], and 50 mM sodium phosphate, pH 7.0) and 10 mL SD20 (20%
dextran sulfate in HB50).
1461
HQ camera (Roper) driven by OpenLAB 4.0.4 software or a Zeiss camera
AxioCam MR driven by Axiovision 4.7. All images were further processed
with OpenLAB 4.0.4, Axiovision 4.7, or Adobe Photoshop 7.0 (Adobe
Systems).
Electron Microscopy
The synaptonemal complex spreads were prepared according to the
protocol described by López et al. (2008) with minor modifications: 0.05%
(v/v) Triton X-100 + 0.1% (v/v) Lipsol detergent was included in the swelling
medium. The slides were stained with aqueous AgNO3 (40%, w/v) at 45°C
and examined under an electron microscope (Jeol 1010). Micrographs were
composed using Photoshop CS4 software (Adobe Systems). A total of 46
and 35 nuclei from allohaploid and 59 and 43 from euploids, respectively,
were photographed (captured nuclei); the remaining nuclei exhibited basically the same features as those selected. A complete reconstruction of
allohaploid captured nuclei was not possible because the axial/lateral elements were partially destroyed in the asynaptic regions.
Statistical Analysis
Student’s t test and x2 analyses were performed using the PROC FREQ
and PROC TTEST procedure of SAS, respectively (SAS Institute Inc.,
1999).
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL
databases under the following accession numbers: KBr036M17,
AC189325; KBrB052L10, AC189386; and KbrH117M18, AC146875.
In Situ Hybridization
Supplemental Data
The probe DNA was prepared by adding ;100 ng each labeled BAC to 9 mL
HB50 and 9 mL SD20. The probe DNA was then denatured at 92°C for 6 min
and stored at 70°C until use. One hundred microliters of RNase was added
to the meiotic spreads and incubated at 37°C for 60 min. Slides were then
rinsed in 23 SSC at room temperature for 2 3 5 min. All washing steps were
performed in Coplin jars. If chromosomes/nuclei appeared to be covered
by cytoplasm after checking slides in a phase contrast microscope, 100 mL
pepsin (100 µg/mL in 0.01 N HCl) was added and the slide was incubated at
42°C until cytoplasm was no longer visible. Care was taken not to treat the
slide with pepsin for too long, to avoid degradation of chromatin structures.
The slides were then postfixed in 4% formaldehyde in 13 PBS at room
temperature for 10 min, rinsed in 23 SSC at room temperature twice for
5 min each, dehydrated through an ethanol series (70, 90, and 100%) for
1 to 3 min each, and air-dried overnight. Then, 100 mL 70% formamide was
added and slides were heated at 70°C for 2 min. After a maximum of 2 h of
air-drying, the slides were dehydrated with a cold ethanol series (70, 90, and
100%) for 1 min each.
Finally, the denaturated probe DNA was added on the slides, which
were then covered with a 22 3 22 cover slip. The slides were placed in
a moist chamber and incubated overnight at 55°C.
The following materials are available in the online version of this article.
Fluorescence Detection
Supplemental Figure 1. Male Meiotic Products in Brassica napus
Euploids.
Supplemental Figure 2. Male Meiotic Products in Brassica napus
Allohaploids.
Supplemental Figure 3. Meiotic Spindle Orientation at Metaphase II in
Brassica napus Allohaploids.
Supplemental Figure 4. Sister Chromatid Cohesion and Meiotic
Chromosome Axes in Brassica napus Euploids.
Supplemental Figure 5. Sister Chromatid Cohesion and Meiotic
Chromosome Axes in Brassica napus Allohaploids.
Supplemental Figure 6. Chromosome Shape and ASY1 Staining
Allow Staging of Prophase I Pollen Mother Cells in Allohaploids.
Supplemental Figure 7. Electron Micrographs of Silver-Stained
Pachytene Nuclei in Brassica napus Euploids.
Supplemental Figure 8. Synaptonemal Complex Formation in Brassica napus Allohaploids Pollen Mother Cells at Pachytene.
Supplemental Figure 9. Progression of Class I Crossover Formation
in Yudal Euploids.
Detection of hybridized probes was performed as described by Lysak
et al. (2006).
Supplemental Figure 10. Immunolocalization of HEI10 and ASY1 in
Darmor-bzh Euploids in Diplotene Pollen Mother Cells Showing
a Multitude of HEI10 Foci along Chromosome Axes.
Fluorescence Microscopy
Supplemental Figure 11. Progression of Class I Crossover Formation
in Yudal Allohaploids.
Observations were made using a Leica DM RXA2 microscope or a Zeiss
Axio Imager 2 microscope; photographs were taken using a CoolSNAP
Supplemental Figure 12. Distribution of the Number of MLH1 Foci per
Cell in Euploid and Allohaploid Brassica napus.
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Supplemental Figure 13. BAC FISH Survey in Brassica napus
Allohaploids: Meiotic Crossover Formation between Homoeologous
Chromosome Pairs A10/C9, A3/C3, and A7/C6, Respectively.
Supplemental Table 1. Distribution of MLH1/HEI10 Foci per Bivalent
in Brassica napus Euploids.
ACKNOWLEDGMENTS
We thank Fabien Nogué, Christine Mézard, and Raphaël Mercier for
critical reading and discussion of the article. We also thank the two
anonymous reviewers for their constructive comments, which helped us
to improve the article. Leigh Gebbie is acknowledged for English
corrections and I. Bancroft for providing the BAC cultures. L.G. was
funded by a PhD fellowship from the French “Ministere de l’Enseignement Superieur et de la Recherche.” N.C. was partially supported by the
Ministerio de Ciencia e Innovación of Spain (Grant BFU2008-00459/
BMC).
AUTHOR CONTRIBUTIONS
L.G., L.C., and E.J. designed the research. L.G., N.C., O.C., V.H., F.E.,
and L.C. performed the research. L.G., N.C., A.M.C., M.G., L.C., and E.J.
analyzed the data. L.G., L.C., and E.J. wrote the article.
Received January 10, 2014; revised March 4, 2014; accepted March 26,
2014; published April 15, 2014.
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Homoeologous Chromosome Sorting and Progression of Meiotic Recombination in Brassica napus:
Ploidy Does Matter!
Laurie Grandont, Nieves Cuñado, Olivier Coriton, Virgine Huteau, Frédérique Eber, Anne Marie
Chèvre, Mathilde Grelon, Liudmila Chelysheva and Eric Jenczewski
Plant Cell 2014;26;1448-1463; originally published online April 15, 2014;
DOI 10.1105/tpc.114.122788
This information is current as of June 17, 2017
Supplemental Data
/content/suppl/2014/04/02/tpc.114.122788.DC1.html
References
This article cites 53 articles, 18 of which can be accessed free at:
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