Homoeologous Chromosome Sorting and Progression ofMeiotic Recombination in Brassica napus: PloidyDoes 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, Franceb AgroParisTech, Institut Jean-Pierre Bourgin, F-78000 Versailles, FrancecDepartamento de Génetica, Facultad de Biología, Universidad Complutense, 28040 Madrid, Spaind INRA, UMR 1349, Institut de Génétique, Environnement et Protection des Plantes, F-35653 Le Rheu Cedex, France

Meiotic recombination is the fundamental process that produces balanced gametes and generates diversity withinspecies. For successful meiosis, crossovers must form between homologous chromosomes. This condition is moredifficult to fulfill in allopolyploid species, which have more than two sets of related chromosomes (homoeologs). Here, weinvestigated 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 resultsdemonstrate a precocious and efficient sorting of homologous versus homoeologous chromosomes during early prophaseI in two representative B. napus accessions that otherwise show a genotypic difference in the progression of homologousrecombination. More strikingly, our detailed comparison of meiosis in near isogenic allohaploid and euploid plants showedthat the mechanism(s) promoting efficient chromosome sorting in euploids is adjusted to promote crossover formationbetween homoeologs in allohaploids. This suggests that, in contrast to other polyploid species, chromosome sorting iscontext dependent in B. napus.

INTRODUCTION

Meiosis is essential to the life cycle of all sexual eukaryotes; thisspecialized type of cell division not only ensures fertility andgenome stability throughout sexual life cycles, but also gen-erates diversity within species by creating new chromosome/allele combinations. All these outcomes depend on the forma-tion of meiotic crossovers (COs), one of the products of meioticdouble-strand break repair occurring during prophase I. At leasttwo classes of COs coexist in plants (Mézard et al., 2007). Thefirst class (class I COs) is dependent on the ZMM (for Zip, Msh,Mer) group of proteins (Osman et al., 2011) and gives rise tointerfering COs, which are further apart from each other thanwould be expected if they were independent. The second class(class II COs) results in noninterfering COs and requires theMETHYL METHANESULFONATE and UV SENSITIVE81 andMETHYL METHANESULFONATE SENSITIVE proteins (Osmanet al., 2011). Due to their role as a physical link between chromo-somes, at least one obligate CO is required between pairs of ho-mologs (Jones 1984), which probably arise from the class Ipathway (Chelysheva et al., 2010). When this obligate CO is abol-ished, chromosomes segregate randomly, resulting in aneuploid

gametes, aneuploid progenies, and reduced fertility (Martinez-Perez and Colaiácovo, 2009). The same negative outcome occurswhen COs are formed between multiple and/or nonhomologouschromosomes, a situation that is more likely to occur in polyploidspecies (reviewed in Grandont et al., 2013).In polyploid species, every chromosome has more than one

potential partner, which can either be identical (when the poly-ploid results from the doubling of a single diploid genome, i.e.,autopolyploidy) or slightly more divergent (when the polyploid hasan interspecific hybrid origin, i.e., allopolyploidy). In any case, thedifferent sets of chromosomes of a polyploid species must besorted out during meiosis to produce balanced gametes. Asa consequence, most allopolyploid species have inherited orevolved recombination-modifying loci that suppress CO forma-tion between the “homoeologous chromosomes” inherited fromthe different parental species (Grandont et al., 2013, and refer-ences therein). The most well characterized of these loci is Pairinghomeologous1 (Ph1) from wheat (Triticum aestivum; Griffithset al., 2006), which was shown to affect CO formation both be-tween homoeologs (Riley and Chapman, 1958; Greer et al.,2012, and references therein) and homologs (Lukaszewski andKopecký, 2010).In the past 10 years, Brassica napus has emerged as another

model for studying natural variation in CO frequency between eitherhomoeologs or homologs. B. napus (AACC, 2n=38) is a recentallotetraploid species that originated from multiple interspecifichybridization events between ancestors of Brassica oleracea (CC,2n=18) and Brassica rapa (AA, 2n=20) (Allender and King, 2010, andreferences therein). These two parental species share an ancestral

1 Address correspondence to eric.jenczewski@versailles.inra.fr.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Eric Jenczewski (eric.jenczewski@versailles.inra.fr).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.114.122788

The Plant Cell, Vol. 26: 1448–1463, April 2014, www.plantcell.org ã 2014 American Society of Plant Biologists. All rights reserved.

whole-genome triplication that occurred soon after the divergenceof the Brassica and Arabidopsis thaliana lineages (Lysak et al.,2005). It has long been known that B. napus has a diploid-likemeiotic behavior, although a small number of homoeologous ex-changes, usually referred to as translocations, have been detectedin 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 createdsynthetic B. napus allotetraploids (i.e., colchicine-doubled B. rapa 3B. 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 homoeologouschromosomes. B. napus allohaploids also show significant variationin meiotic behavior at metaphase I, with two main meiotic pheno-types identified across a wide range of varieties (Nicolas et al., 2009;Cifuentes et al., 2010, and references therein). This variation relies onpolygenic control, with one major quantitative trait locus, namedPairing regulator in Brassica napus (PrBn), being far more influentialthan the others (Cifuentes et al., 2010, and references therein).

Our current understanding of CO variation in B. napus is mainlybased on cytological observations at metaphase I and geneticsurveys of intergenomic exchanges in the progenies of B. napusallohaploids. As these approaches focused on the final products ofmeiotic recombination (chiasmata and genetic exchanges), they didnot provide comprehensive information on the way(s) homologousbivalents form in euploid B. napus or the causes for the observeddifference in CO rate between allohaploid plants. We do not evenknow whether the same strategy for homologous bivalent forma-tion is implemented in B. napus euploid accessions originating fromdifferent polyploidization events (Cifuentes et al., 2010). The aim ofthis study, therefore, was to specifically address these questions.For this, we set up a thorough comparative analysis of meiosis ineuploid and allohaploid plants chosen to be representative of (1) thedifferent origins of B. napus and (2) the dichotomy of meiotic phe-notypes observed among B. napus allohaploids. We investigatedthe formation, progression, and completion of several key hallmarksof meiosis, including sister chromatid cohesion, chromosome axes,the synaptonemal complex, meiotic recombination, in order to de-termine the extent to which these factors contribute to the observeddifferences or vary with the different origins of B. napus. Altogether,our results demonstrate that homologous and homoeologouschromosomes are sorted at an early stage of prophase I in two B.napus genotypes, which otherwise show varying numbers of class ICOs. However, and contrary to other polyploid species, themechanisms regulating efficient chromosome sorting in euploids donot suppress CO formation between homoeologs in the near iso-genic allohaploids.

RESULTS

B. napus Euploids Show a Diploid-Like Meiotic Behavior

We first compared progression of male meiosis in Darmor-bzhand Yudal euploid genotypes (with 2n=38 chromosomes, i.e.,AACC), which are representative of the two main B. napus genepools (Harper et al., 2012). Staining chromosomes with 49,6-diamidino-2-phenylindole (DAPI) showed that meiosis is veryregular in Darmor-bzh and Yudal euploids (Figure 1) and very

similar to meiosis in Arabidopsis (Ross et al., 1996). Briefly, meioticchromosomes first condensed at leptotene (Figures 1A and 1K). Atzygotene, we observed several close alignments of chromosomes(Figures 1B and 1L), which are diagnostic for the establishment ofthe synaptonemal complex. At pachytene, all chromosomes wereclosely aligned with one another (Figures 1C and 1M), indicatingthat homologous chromosomes were fully synapsed (see below).The synaptonemal complex then disassembled and chromosomescondensed until discrete separate bivalents became visible atdiakinesis (Figures 1D and 1N). At metaphase I, we always ob-served 19 bivalents in the two genotypes with readily discerniblechiasmata, the cytological manifestation of meiotic COs (Figures 1Eand 1O). Based on chromosome shape (Jones 1984), we estimateda mean cell chiasmata frequency (n = 20 pollen mother cells[PMCs]) of 35 in both Darmor-bzh and Yudal (Table 1). The secondmeiotic division then took place (Figures 1G to 1I and 1Q to 1S). Itresulted in four balanced sets of 19 chromatids (Figures 1J and 1T)that invariably led to tetrad formation (n = 250 and 251 cells forDarmor-bzh and Yudal euploids, respectively; Supplemental Figure1). In both Darmor-bzh and Yudal, pollen grains were 100% viable(Supplemental Figure 1), demonstrating that meiosis in B. napuseuploids leads to faithful chromosome segregation.

B. napus Allohaploids Show Impaired Meiosis but StillProduce a Few Viable Pollen Grains

The early stages of meiosis in allohaploid plants (with 19 chromo-somes, i.e., AC) appeared similar to those described in euploids.The first noticeable difference occurred at pachytene (Figures 2Aand 2J). Both unaligned regions and stretches of juxtaposedregions between pairs of nonhomologous chromosomes wereobserved in all PMCs at this stage (hereafter referred to aspachytene-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 theywere often entangled, especially in Yudal allohaploids. At meta-phase I (Figures 2C and 2L), both univalents (i.e., chromosomesthat failed to form chiasmata) and bivalents were observed, withvariable numbers between genotypes. The number of chiasmatawas also significantly higher in Darmor-bzh (9.25 on average) thanin Yudal (5.70 on average) allohaploids (P = 0.0001; Table 1),which is consistent with Cifuentes et al. (2010). In contrast to ourobservations in euploids, these chiasmata were formed betweennonhomologous A and C chromosomes because allohaploids donot contain homologous chromosomes.Regardless of the number of chiasmata formed, in allohaploid

telophase I nuclei (Figures 2E and 2N), the chromosome com-position was unequal, which, in most cases, led to unbalancedtetrads (Figures 2H, 2Q, and 2R) or polyads (when laggardchromosomes lead to the formation of additional nuclei) and,thus, to unviable pollen grains (Supplemental Figure 2). Un-expectedly, meiosis in Darmor-bzh and Yudal allohaploids alsoproduced varying numbers of triads (Figure 2I) and dyads (Figure2S); these were the minority in Darmor-bzh allohaploids (;5%),but they represented half the products formed during meiosis inYudal allohaploids (Supplemental Figure 2). Immunolocalization ofa-tubulin (a component of the meiotic spindle) showed that the

Meiotic Crossovers in Brassica napus 1449

dyads and triads resulted from the formation of parallel and/ortripolar spindles during metaphase II (Supplemental Figure 3),which regrouped the products of the first division. Thus, the re-sulting microspores contained the 19 chromosomes of the basicB. napus chromosome set and probably generated the viablepollen grains observed in the two allohaploid genotypes (seepollen colored in red in Supplemental Figure 2).

Chromosome Axes Are Correctly Formed in Both Euploidand Allohaploid B. napus

Next, we investigated sister chromatid cohesion and assemblyof meiosis-specific chromosome axes in B. napus, as these twomeiotic landmarks are essential for CO formation. For this, we

coimmunolocalized themeiosis-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 allo-

haploid B. napus (Supplemental Figures 4 and 5). The two proteinswere correctly loaded on chromatin at leptotene and progressivelyformed a continuous signal as the chromosomes condensed andsynapsed. The two signals colocalized along the entire length of thechromosome axes, whether they were synapsed or not in pachy-tene or pachytene-like cells. The signals persisted at diakinesis andmetaphase I in both allohaploid and euploid B. napus.These results suggest that sister chromatin cohesion and

chromosome axes are correctly established and maintained in

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 condenseand become visible as unpaired threads. Zygotene ([B] and [L]): Arrows indicate several close juxtapositions of chromosomes that probably mark the initiationof the synaptonemal complex. Pachytene ([C] and [M]): All chromosomes are closely aligned with one another, suggesting that the synaptonemal complex iscomplete. Diakinesis ([D] and [N]): chromosomes are condensed and form discrete separate bivalents. Metaphase I ([E] and [O]): All bivalents aligned on themetaphase plate. Anaphase I ([F] and [P]): Homologous chromosomes, each composed of two sister chromatids, move to the opposite poles. Chromosomebridges 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.

1450 The Plant Cell

the two genotypes. As no difference was observed betweenDarmor-bzh and Yudal, the difference in meiotic behaviors ob-served at metaphase I in allohaploids is not due to an obvious de-fect in chromosome axis formation. Chromosome shape (based onDAPI staining) and ASY1 staining were also instrumental fordiscriminating between the different prophase I-like stages inallohaploids (Supplemental Figure 6). The chromosomes in earlyzygotene PMCs had a compact appearance and were stronglylabeled with the anti-ASY1 antibody, whereas in pachytene-likePMCs, chromosomes looked less compact and a continuous ASY1signal was observed. The chromatin in late diplotene PMCs wasvery diffuse and the ASY1 signal began to disappear from thechromosome axes. These criteria were used in the rest of our study.

The Extent and Nature of Synapsis Is Different in B. napusEuploids and Allohaploids

We then used two complementary approaches to gain a betterunderstanding of synaptonemal complex formation in B. napuseuploids (Figure 3). First, to monitor the progression of synapsis, weimmunolocalized ZIPPER1 (ZYP1), a component of the transversefilament of the synaptonemal complex in A. thaliana (Higgins et al.,2005). Second, we analyzed surface-spread prophase I nuclei withelectron microscopy to examine whether synaptic multivalents areformed prior to or at pachytene.

The ZYP1 signal overlapped with the ASY1 signal along theentire length of the chromosomes in both Darmor-bzh and Yudaleuploids (Figures 3A and 3B). This indicates that full synapsis doesoccur at pachytene in the two genotypes (n = 15 and n = 18 PMCs,respectively), even if the signal in Yudalwas never as continuous asin Darmor-bzh. Electron microscopy observations confirmed thatsynapsis is complete in the two genotypes with no apparent dif-ference between them (59 and 43 nuclei from late zygotene topachytene were reconstructed for Darmor-bzh and Yudal euploids,respectively). In the two genotypes, 19 synapsed bivalents, whichmost likely correspond to homologous pairs, were observed only in;50% of nuclei (Figures 3C and 3D, Table 2). In the other;50% ofnuclei, one (40% of nuclei) or two (;10% of nuclei) synaptic

quadrivalents, formed by the association of four chromosomesjoined at different points, were observed, with no differencebetween the two genotypes (Figures 3E to 3G, Table 2;Supplemental Figure 7). The majority of these synaptic quad-rivalents had only one synaptic partner switch (Figures 3E and3G), and only 15% had two synaptic partner switches (Figure3F). Most synaptic partner switches were preferentially locatednear the chromosome ends (subterminal or distal position),while a few occurred in more interstitial regions. We neverobserved a synaptic partner switch within the central part ofthe chromosomes. When synaptonemal complexes weremeasured in nuclei showing no or little distortion using electronmicroscopy (i.e., no interlock, no synaptonemal complexfragmentation, and no foldback loop), no significant differencein synaptonemal complex length between Darmor-bzh andYudal 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 forthe euploids (Supplemental Figure 8). This confirmed that synapsisis never complete in allohaploids. Although several continuousstretches of ZYP1 signal were observed in both Darmor-bzh andYudal allohaploids, most of these tracts were shorter than thechromosome length and were systematically accompanied byunsynapsed chromosome axes labeled by ASY1, but not ZYP1 (n =16 and n = 15 PMCs, respectively). Nevertheless, electron mi-croscopy did reveal the presence of one or two completely syn-apsed bivalents (Supplemental Figure 8) in ;50% of prophase Inuclei, amid partially synapsed or completely unsynapsed chro-mosomes. These complete synaptic bivalents appeared to be asfrequent in Darmor-bzh as in Yudal allohaploids (n = 46 and n = 35PMCs, respectively).The absence of complete synapsis made comparison be-

tween Darmor-bzh and Yudal allohaploids difficult; cell-to-celldifferences could reflect either differences in the extent of syn-aptonemal complex completion or differences in the stages thatwere observed. With this limitation in mind, no obvious differ-ence in synaptic behavior was found between Darmor-bzh and

Table 1. Progression of Meiotic Recombination in B. napus Euploids (AACC; 2n=38) and Allohaploids (AC; n = 19)

PMCs at Pachytene PMCs at Diplotene PMCs at Diakinesis PMCs at Metaphase I

GenotypesNo. ofPMCs

Avg. No. ofHEI10 Focia

No. ofPMCs

Avg. No. ofHEI10 Foci

No. ofPMCs

Avg. No. ofMLH1 Foci

Avg. No. of HEI10+ MLH1 Foci

No. ofPMCs

Avg. No. ofBivalents

Avg. No. ofChiasmata

Darmor-bzheuploids

13 27.46 (16–36) 7 23.86 (20–28) 33 – 34.82 (25–45) 20 19 35.45

Yudaleuploids

37 28.05 (18–38) 11 29.09 (25–36) 29 – 31.00 (21–38) 20 19 35.05

n.s. n.s. **Darmor-bzh

allohaploids25 24.20 (14–40) – – 207 18.34 (3–39) 6.24 (1–10)b 19 6.67 (5–8) 8.92

Yudalallohaploids

71 16.41 (7–28) – – 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).aThese values were obtained for PMCs showing only distinct HEI10 foci.bThese values were obtained for 17 and 30 PMCs for Darmor-bzh and Yudal allohaploids, respectively.

Meiotic Crossovers in Brassica napus 1451

Yudal allohaploids with either immunolocalization or electronmicroscopy.

The Dynamics of Class I CO Formation Is Differentbetween the Two Genotypes

We monitored the progression of meiotic recombination byimmunostaining HUMAN ENHANCER OF INVASION10 (HEI10),a key protein that is unique because it allows the progressivetransition from early recombination intermediates into final class ICOs to be scrutinized (Chelysheva et al., 2012).

HEI10 showed the same dynamic localization pattern duringprophase 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 (Figure4A; Supplemental Figure 9A). During pachytene, the number ofHEI10 foci dropped dramatically, changing from a zygotene-likepattern (Figure 4B; Supplemental Figure 9B) until only a few dis-tinct foci persisted on chromatin (Figure 4C; Supplemental Figure9C). At diplotene (Figure 4D; Supplemental Figure 9D), distinctHEI10 foci were still present on the chromosomes where theysystematically 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 inB. napus euploids is progressively restricted to the sites whereclass I COs mature.

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 ofchromosomes 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, eachcomposed of two sister chromatids, are separated. At this stage chromosome bridges are often observed as in euploids. Telophase I ([E] and [N]): Twogroups 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 unbalancedtetrads ([H], [Q], and [R]), triads (I), as well as dyads (S). Bars = 5 mm.

1452 The Plant Cell

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 synapticbivalents. Bars = 5 mm.(E) to (G)High-magnification electronmicrographs of quadrivalents in pachytene nuclei ofDarmor-bzh ([E] and [F]) andYudal (G) euploidswith 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.

Meiotic Crossovers in Brassica napus 1453

This HEI10 dynamic pattern was observed in both Darmor-bzhand Yudal euploids. However, two important differences werefound. First, the pronounced decline in HEI10 foci was evenstronger in Yudal than in Darmor-bzh (Figure 5). In Darmor-bzheuploids, 82% (60 out of 73) and 46% (6 out of 13) PMCs stillshowed numerous HEI10 foci along chromosome axes atpachytene and diplotene, respectively (Figure 5B; SupplementalFigure 10). By contrast, in Yudal euploids, the vast majority ofPMCs (95% at pachytene, 37 out of 39; 100% at diplotene)showed only distinct HEI10 foci (Figure 5G). No significant dif-ference was found when the number of HEI10 foci was comparedin Darmor-bzh and Yudal pachytene PMCs that showed onlydistinct HEI10 foci (Student’s t test; P = 0.7); only a moderatedifference was found at diplotene (P = 0.007).

Second, the mean number of foci where HEI10 and MLH1colocalized 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’st test; P = 0.0014), suggesting that Darmor-bzh and Yudal differslightly 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 ofmeiotic recombination in Darmor-bzh and Yudal allohaploids(Figure 6; Supplemental Figure 11) and observed essentially thesame 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 thelate HEI10 pattern were seen in 90% (71 out of 79) of pachytene-like PMCs, whereas these were observed in only 25% (25 out of98) of PMCs in Darmor-bzh allohaploids. The largest HEI10 fociobserved in B. napus allohaploids were never found on un-synapsed regions even at the end of pachytene (Figure 6C;Supplemental Figure 11C). This suggests that the formation ofdistinct HEI10 foci occurs only on synapsed regions.

Despite these similarities, we found three differences betweeneuploids and allohaploids. First, in allohaploids but not euploids,the mean number of HEI10 foci in pachytene-like PMCs wassignificantly higher in Darmor-bzh than in Yudal (24.2 versus 16.4;P < 0.001). This was mainly due to a significant decrease in thenumber of HEI10 foci in Yudal allohaploids compared with Yudaleuploids (Table 1; P < 0.001), whereas the estimates were thesame in Darmor-bzh allohaploids and euploids (Table 1; P = 0.19).

Second, almost twice as many MLH1 foci were seen inDarmor-bzh than Yudal allohaploids (on average, 18 versus 10foci, respectively; Student’s t test, P < 0.001; Table 1, Figure 6E;Supplemental Figure 11E), while the variation was much smallerbetween euploids. Indeed, the distribution of MLH1 foci per cellfit a Poisson distribution in the two allohaploids but not in theeuploids, in which an excess of counts near the mode and largedeficiencies at the extremes of the distribution were found(Supplemental Figure 12). This strongly suggests that the dis-tribution of MLH1 foci was random between PMCs in allo-haploids, while it was constrained in euploids; this also explainswhy data counts were more widely dispersed in allohaploidsthan in euploids (Table 1).Finally, coimmunolocalization of MLH1 with HEI10 revealed

that the two proteins only rarely colocalized at diakinesis in allo-haploids (Figure 6F), but systematically did so in the corre-sponding euploids (Figure 4E; Supplemental Figure 9E). Distinctand separate MLH1 and HEI10 foci were repeatedly observed atdiakinesis in three independent experiments, in both Darmor-bzhand Yudal allohaploids. While the identity of the recombinationintermediates marked by the distinct and separate MLH1 andHEI10 foci remain unknown, the two proteins colocalized in morefoci in Darmor-bzh than in Yudal allohaploids (6.2 versus 2.5; P <0.001). Thus, a significant difference was found between the twoallohaploids regardless of which criteria were used to assessclass 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- andGenotype-Specific Effects

Finally, we characterized recombination between individual chro-mosomes during meiosis in allohaploid B. napus. For this, BACfluorescence in situ hybridization (FISH) experiments were per-formed using some of the BAC clones identified by Xiong et al.(2010), which specifically and simultaneously label pairs of ho-moeologous chromosomes/regions. We first focused on the A1-C1pair of homoeologs (Figure 7), which are collinear along their entirelength (Parkin et al., 2005). A1 and C1 were frequently held togetherby 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 boundthe two arms (thus, at least two COs) of a slightly higher proportionof bivalents than in Yudal allohaploids (72.3% versus 60.4%). In thefew PMCs in which A1 and C1 did not recombine together atmetaphase I in Darmor-bzh (10 PMCs out of 129; 7.8%) or Yudal(nine PMCs out of 57; 15.8%) allohaploids, the chromosomesformed either a bivalent with another chromosome (Figure 7C) orremained 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 longarm of A10/short arm of C9 (Parkin et al., 2003, 2005; Xiong et al.,2010). Every pair showed a distinct and specific pattern at meta-phase I (Supplemental Figure 13): A10 and C9 recombined togethermuch more frequently in Darmor-bzh than in Yudal allohaploids(Table 3; P = 0.0095); A7 and C6 recombined together in the sameproportion of PMCs in Darmor-bzh and Yudal allohaploids (Table 3;

Table 2. Synaptic Associations in B. napus Euploids (AACC; 2n=38) atLate Zygotene-Pachytene

Number of PMCs withTotal No.of PMCs

Genotypes 19 II 17 II + 1 IV 15 II + 2 IV

Darmor-bzheuploids

28 (47.5%) 24 (40.7%) 7 (11.8%) 59

Yudal euploids 19 (44.2%) 20 (46.5%) 4 (9.3%) 43

II, bivalents; IV, quadrivalents. Values in parentheses indicate theproportion of cells in the different classes.

1454 The Plant Cell

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.

Meiotic Crossovers in Brassica napus 1455

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 distinctHEI10 foci on chromosome axes (in orange) is shown for Darmor-bzh euploid ([A] to [D]) and Yudal euploid ([E] to [H]). Bars = 5µm.

1456 The Plant Cell

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).

P = 0.7512); A3 and C3 recombined together a little more often inYudal than in Darmor-bzh allohaploids (Table 3; P = 0.1915). Wewere also able to distinguish a series of other bivalents, for whichonly one operating chromosome was identified by the probes weused (e.g., A3-Cx, C9-Ax.). These additional bivalents morefrequently involved A3 and/or C3 in Darmor-bzh allohaploidsand A10 and/or C9 in Yudal allohaploids. This suggests thatchromosome- and genotype-specific factors can change theodds of forming a CO between a given pair of homoeologousregions irrespective of the overall difference in CO rate betweenDarmor-bzh and Yudal allohaploids.

DISCUSSION

Early Homoeologous Chromosome Sorting inB. napus Euploids

Cytological diploidization of allopolyploid species can beachieved in different ways, depending on the stage when ho-mologous and homoeologous chromosomes are successfullysplit apart (Jenkins and Rees, 1991). In this study, we showedthat homoeologous chromosomes are sorted early on duringprophase I in B. napus, synapsis being mostly restricted tohomologs by late zygotene-pachytene in both Darmor-bzh andYudal euploids. We observed no more than one or two syn-aptic multivalents in half of the PMCs observed at these stages(Table 2), the origin of which can be interpreted in two differentways.The most straightforward hypothesis is that the synaptic multi-

valents result from synaptonemal complex formation betweenhomoeologous regions. In that case, B. napus would be like wheat,Aegilops, and Festuca polyploids, in which varying numbers ofhomologous and homoeologous chromosomes are partially syn-apsed during prophase I (Hobolth, 1981; Thomas and Thomas,1993; Cuñado et al., 1996). Given that, in plants, the synaptonemalcomplex does not form when strand invasion is impaired (Osmanet al., 2011), the presence of homoeologous synaptic associationsmost likely indicates that strand exchanges are initiated betweenhomoeologs in B. napus euploids. This is consistent with thedemonstrated propensity for homoeologous chromosomes to re-combine with one another in allohaploids (Figures 2 and 5). How-ever, given the limited extent to which synaptic multivalentspersisted to pachytene in B. napus euploids, it appears that mostinterhomoeolog recombination intermediates abort early and areprobably redirected into intersister or noncrossover pathways(Hunter and Kleckner, 2001). Evidence for very short nonreciprocalexchanges between homoeologous sequences, which possiblyoriginated from meiotic noncrossovers, was recently obtainedin cotton (Gossypium hirsutum), another bivalent-forming allo-polyploid species (Salmon et al., 2010; Flagel et al., 2012). Furtherstudies are needed to demonstrate that similar events occur in B.napus.As an alternative, the synaptic multivalents observed at pachy-

tene may result from the presence of translocated A/C chromo-some(s), in which one region has been replaced by its homoeologthat is thus duplicated within the genome. In that case, which wasfirst advocated by Osborn et al. (2003), the synaptonemal complexcould be formed only between homologous segments, albeitcarried by different chromosomes; this would mean that B. napus

Figure 7. BAC FISH Survey of CO Formation for a Given Pair ofHomoeologous Chromosomes (A1-C1) in B. napus Allohaploids.

Chromosomes were stained with DAPI (white, false color), and FISH wasperformed using two BACs that specifically and simultaneously identifyA1 and C1 chromosomes (red and green). Metaphase I PMCs showingeither only one bivalent labeled, indicating that A1 and C1 recombinedtogether ([A] and [B]) or showing two labeled entities indicating that A1and C1 did not recombine together ([C] and [D]). In (C), two bivalents arelabeled, indicating that A1 and C1 recombined with other chromosomes(arrows). In (D), two univalents are labeled, indicating that A1 and C1 didnot recombine. Bars = 5 µm.

Figure 6. (continued).

(D) Immunolocalization of HEI10 at diplotene. Chromosomes were labeled with DAPI (white, false color) and HEI10 (green) antibodies. An overlay of thetwo signals is shown (merge).(E) Immunolocalization of MLH1 at diakinesis. Chromosomes were labeled with DAPI (white, false color) and MLH1 (green) antibodies. The overlay oftwo 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 showthe colocalization of MLH1 and HEI10 foci.Bars = 5 mm.

1458 The Plant Cell

homoeologous regions are sorted at an even earlier stage, or evenmore efficiently than envisaged above. This is exactly what hap-pens in oat (Avena sativa), Avena marrocana, and Allium mon-tanum, in which the synaptonemal complexes appear to beconfined to homologous bivalents from zygotene onwards (Loidl,1988; Jones et al., 1989). Although to date no A/C translocationshave been described in Darmor-bzh and Yudal, their existencewould help explain some of our BAC FISH observations; becausethe chance for CO formation is higher between duplicated ho-mologous segments than between homoeologous regions, trans-located A/C chromosome(s) (e.g., presumably A3/C3 in Yudal) areexpected to recombine much more systematically than non-recombinant chromosomes (Table 3). Thus, more work is neededto confirm the presence of translocated A/C chromosome(s) inDarmor-bzh and Yudal and their possible consequence on thesynaptonemal complex.

Regardless of the origin of the synaptic multivalents, our resultssuggest that, in B. napus euploids, homoeologous chromosomesare sorted prior to, or during, formation of stable strand exchanges,which are thought to occur during zygotene concomitant withsynaptonemal complex formation (Hunter and Kleckner, 2001; Tianget al., 2012). The remaining multivalents are then eliminated beforediakinesis, as if a second layer of control suppresses CO formationwithin the “illegitimately” synapsed regions. This latter adjustmentprobably remains an error-prone process and a few COs can beexpected to occasionally form between homoeologs in euploids, inthe regions where synapsis first took place. Our results suggest thatthese are fairly rare events (Table 1), although we have no evidencethat 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 ineuploids is paralleled by an almost complete suppression of COformation between homoeologs in the corresponding allohaploids(Riley and Chapman, 1958; Gauthier and McGinnis, 1968). In theseplants, even if chromosomes have no choice but to recombine withtheir homoeologs, they are prevented from doing so by loci re-sponsible for the cytological diploidization of the euploid forms(Griffiths et al., 2006; Greer et al., 2012, and references therein). Ourresults show that the situation is strikingly different in B. napus; thehomoeologous synaptic and chiasmatic associations that are sup-pressed in euploids become dominant in allohaploids. This indicatesthat the mechanism(s) responsible for the early sorting of homo-eologous chromosomes in euploid B. napus do(es) not suppressCO formation between homoeologous chromosomes in allo-haploids. This does not mean, however, that there is no sortingprocess operating in B. napus allohaploids. On the contrary, COs

are preferentially formed between homoeologs in these plants andmore occasionally between the duplicated blocks inherited from theBrassica ancestral whole-genome triplication (i.e., paleologs)(Nicolas et al., 2009, and references therein). Thus, it is as if a flexiblebut efficient sorting mechanism is adjusted to promote CO forma-tion between the closest available matches (homologs in euploidsand homoeologs in allohaploids), disregarding the other potentialpartners (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 ofchromosomes 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 isset to become mature, a decision that cannot be made only locally(e.g., based on the stability of DNA heteroduplexes) because oth-erwise the intermediates that are committed to form COs in allo-haploids could hardly be aborted in euploids. Instead, homologyappears to be processed along the entire chromosome length. Thisdestabilizes the weakest or less common nascent recombinationintermediates formed between the most divergent chromosomesand promotes the strongest or more numerous intermediates thatwere formed, from the outset, between the least divergent chro-mosomes. As the outcome of this competition depends on theoperating chromosomes, the most likely winners are expected tochange with context.The mechanism(s) for this “context-dependent chromosome

sorting” is (are) unknown but appear(s) to be as efficient or workwith almost the same level of stringency in Darmor-bzh andYudal (see below). Indeed, no clear difference was observedbetween the two genotypes, which showed almost the sameproportion of pachytene PMCs with 19 synapsed bivalents andthe same frequency of synaptic multivalents (Table 2). Why,then, are there different numbers of meiotic COs between ho-moeologous chromosomes in allohaploid plants (Table 1)?

Variation in Class I CO Numbers betweenB. napus Varieties

In this study, we first demonstrated that the spatial-temporallocalization of HEI10, a ZMM protein essential for CO forma-tion in plants, is the same during prophase I in B. napus eu-ploids as in A. thaliana and rice (Chelysheva et al., 2012; Wanget al., 2012). Thus, as with these two species, the dynamicrelocation of HEI10 in B. napus probably reflects the pro-gressive channeling of recombination intermediates into theZMM CO pathway (see Chelysheva et al., 2012 for details). Wethen showed that this progression varies from one genotypeto another. The transition from early to late HEI10 staining

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 92.8% (119/129) 80% (16/20) 53% (18/34) 40% (8/20)Yudal allohaploids 84.2% (48/57) 35% (7/20) 71.4% (20/28) 50% (10/20)

Values in parentheses indicate the number of PMCs over the total number of PMCs observed.

Meiotic Crossovers in Brassica napus 1459

appeared sharper in Yudal than in Darmor-bzh (Figure 5). Finally,immunolocalization of MLH1 demonstrated that Darmor-bzh andYudal form different numbers of class I COs (Table 1). Although itremains to be established whether the different patterns and/orchronology of HEI10 relocation contribute to this variation in class ICOs, our results suggest that there are more COs in Darmor-bzhthan in Yudal, all things being equal. Does it follow that allohaploidswith higher CO frequencies have originated from allotetraploidaccessions also showing higher numbers of homologous COs?

Interestingly, B. napus allohaploids displayed the samezygotene-to-diplotene patterns of HEI10 staining as the corre-sponding euploids (Figure 6; Supplemental Figure 11). This sug-gests that the progression of early meiotic recombination isessentially the same whether recombination intermediates areformed 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 synapsedregions in allohaploids (Figure 6C; Supplemental Figure 11C),suggesting that these are probably dependent on synaptonemalcomplex formation or conversely required to nucleate synapto-nemal complex (see Reynolds et al. [2013] for comparable resultsin mice). Even more strikingly, we observed that the number ofMLH1 foci per cell fit a Poisson distribution in the two allohaploids,as if they were distributed at random between PMCs. As firstproposed by Jones (1967), this distribution may actually reflecta breakdown of the process(es) regulating the distribution of MLH1foci at the bivalent level. In line with this hypothesis, we observedthat only a small fraction of HEI10 and MLH1 foci colocalized atdiakinesis in B. napus allohaploids (Figure 6F), while this wassystematic in euploids (Figure 4E; Supplemental Figure 9E). Thenature of these separate HEI10 and MLH1 foci is unknown, but it isprobably no coincidence that they were found in plants showingpartial synapsis (Qiao et al., 2012, and references therein). As re-cently proposed for the tomato (Solanum lycopersicum) asynapticmutant as1 (Qiao et al., 2012) or haploid Arabidopsis (Cifuenteset al., 2013), the “stand-alone” MLH1 foci could in fact mark thelocations where COs eventually failed or occurred between sisterchromatids. As there is bias against forming COs between sisterchromatids (at least in haploid Arabidopsis; Cifuentes et al., 2013),it is possible that a fraction of late recombination intermediates arestill in the process of being resolved at diakinesis in B. napusallohaploids, resulting in some HEI10 foci persisting longer thanusual on chromosomes without producing the conditions requiredfor MLH1 loading. Finally, separate HEI10 and MLH1 foci mayreflect an aberrant turnover of meiotic proteins in allohaploidplants, with MLH1 loading and off-loading irrespective of HEI10.

Regardless of the causes and consequences of separate HEI10and MLH1 foci, there were significantly more HEI10 foci, moreMLH1 foci, and more “MLH1+HEI10” foci in Darmor-bzh allo-haploids than Yudal allohaploids (Table 1; Supplemental Figure 12).Variation in class I CO rates may thus explain a significant pro-portion of the variation in total CO rate observed between Darmor-bzh and Yudal allohaploids. It is of note, however, that the meannumber of distinct HEI10 foci is significantly lower at pachytene inYudal allohaploids compared with Yudal euploids (16.4 versus 28.0foci; P < 0.001; Table 3), while it is almost the same in Darmor-bzhallohaploids 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 inYudal allohaploids than in euploids (or in Darmor-bzh). Thus, ourresults point toward a possible difference in the sensitivity tochromosome divergence between the two genotypes.In fact, it may be pointless to consider suppression of COs be-

tween homoeologs and variation in CO number between homologsas separate processes in allopolyploid species. There is some in-dication, for instance, that Ph1, the main locus responsible for thecytological diploidization of wheat (Riley and Chapman, 1958;Griffiths et al., 2006; Greer et al., 2012, and references therein), af-fects recombination between dissimilar homologs (Lukaszewski andKopecký, 2010, and references therein), suggesting that this locusregulates 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 andYudal was described by Cifuentes et al. (2010).The plants were cultivated ina greenhouse or growth chamber under a 16-h-light/8-h-night photoperiod, at22°C day and 18°C night, with 65% humidity.

Cytology

Male meiotic spreads for DAPI staining were prepared as described byChelysheva et al. (2013) from buds fixed in Carnoy’s fixative (absoluteethanol:acetic acid, 3:1, v/v).

Final male meiotic products were observed by toluidine blue stainingas described by Azumi et al. (2002), and mature pollen grain viability wasestimated as described by Alexander (1969). Images were taken witha Leica Diaplan bright-field microscope.

Immunolocalization of a-Tubulin

Anthers dissected from fixed buds at the appropriate meiotic stage werestained according to a protocol adapted from Mercier et al. (2001). Buds wereincubated for 5 min in citrate buffer, pH 6.0, and rinsed in water. After a firstdigestion of 1 h in digestionmix (0.3% [w/v] cellulaseRS, 0.3% [w/v] pectolyaseY23, and 0.3% [w/v] cytohelicase in citrate buffer), one anther was placed onapolysin slide, dissected inwater, squashed, andfixed innitrogen. The releasedcells 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 fora further 1 h in digestion mix at 37°C. After three rinses in 0.1%PBS-T (10 mMsodium phosphate, pH 7.0, 143 mM NaCl, and 0.1% Triton X100), cells wereincubated for 1 h and 30min in 1%PBS-T (10mM sodium phosphate, pH 7.0,143mMNaCl, and 1%Triton X100) at room temperature and rinsed two timeswith 0.1%PBS-T. Cells were incubated overnight at 4°C with an anti-a-tubulinmouse 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 incubatedfor 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 at37°C. After three rinses in 0.1% PBS-T, the cells were mounted in Vectashieldantifade medium (Vector Laboratories) with 2 mg/mL DAPI.

Immunolocalization of Meiotic Proteins

Coimmunolocalization of ASY1/REC8, HEI10/ASY1, and MLH1/HEI10 wasperformed on meiotic spreads prepared as described previously for DAPIstaining. Immunolabeling was performed as described by Chelysheva et al.

1460 The Plant Cell

(2013).ThecoimmunolabelingofASY1/ZYP1wasperformedasdescribedbyChelysheva et al. (2007) from fresh buds. The anti-ASY1 polyclonal antibodyhas been described elsewhere (Armstrong et al., 2002). It was used at a di-lutionof 1:250.The anti-REC8polyclonal antibody (Cai et al., 2003)wasusedat 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 (Chelyshevaet al., 2012) was used at a dilution of 1:20 (purified serum). The anti-ZYP1polyclonal antibody (Higgins et al., 2004) was used at a dilution of 1:500.

BAC FISH Experiment

BAC FISH experiments onmeiotic spreads (see above) were performed asdescribed 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) andexpected to hybridize to homoeologous regions of the C genome of B.napus (Xiong and Pires, 2011) were used. The five BACs were labeled usingBiotin-Nick TranslationMix (Roche) andDIGNick TranslationMix (Roche) asdescribed by Lysak et al. (2006) or random priming with Alexa 488-5-dUTPand biotin-14-dUTP (Invitrogen, Life Technologies) (Suay et al., 2013). Thelabeled BACs were then precipitated and the dry pellet dissolved in 10 mLHB50 (50% deionized formamide, 23 SSC [0.30 mM NaCl and 0.030 MNa3-citrate], and 50mM sodium phosphate, pH 7.0) and 10 mL SD20 (20%dextran sulfate in HB50).

In Situ Hybridization

TheprobeDNAwaspreparedbyadding;100ngeach labeledBAC to9mLHB50and9mLSD20. TheprobeDNAwas thendenatured at 92°C for 6minand stored at 70°C until use. One hundredmicroliters of RNasewas addedto the meiotic spreads and incubated at 37°C for 60 min. Slides were thenrinsed in23SSCat roomtemperature for 235min.Allwashingstepswereperformed in Coplin jars. If chromosomes/nuclei appeared to be coveredby cytoplasmafter checking slides in aphase contrastmicroscope, 100mLpepsin (100 µg/mL in 0.01 NHCl) was added and the slidewas incubated at42°C until cytoplasmwas no longer visible. Care was taken not to treat theslidewithpepsin for too long, to avoiddegradationof chromatin structures.The slides were then postfixed in 4% formaldehyde in 13 PBS at roomtemperature for 10 min, rinsed in 23 SSC at room temperature twice for5 min each, dehydrated through an ethanol series (70, 90, and 100%) for1 to 3min each, and air-dried overnight. Then, 100mL 70% formamidewasadded and slides were heated at 70°C for 2 min. After a maximum of 2 h ofair-drying, theslidesweredehydratedwithacoldethanolseries (70,90,and100%) for 1 min each.

Finally, the denaturated probe DNA was added on the slides, whichwere then covered with a 22 3 22 cover slip. The slides were placed ina moist chamber and incubated overnight at 55°C.

Fluorescence Detection

Detection of hybridized probes was performed as described by Lysaket al. (2006).

Fluorescence Microscopy

Observations were made using a Leica DM RXA2 microscope or a ZeissAxio Imager 2 microscope; photographs were taken using a CoolSNAP

HQ camera (Roper) driven by OpenLAB 4.0.4 software or a Zeiss cameraAxioCam MR driven by Axiovision 4.7. All images were further processedwith OpenLAB 4.0.4, Axiovision 4.7, or Adobe Photoshop 7.0 (AdobeSystems).

Electron Microscopy

The synaptonemal complex spreads were prepared according to theprotocol 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 swellingmedium. The slides were stained with aqueous AgNO3 (40%, w/v) at 45°Cand examined under an electronmicroscope (Jeol 1010). Micrographs werecomposed using Photoshop CS4 software (Adobe Systems). A total of 46and 35 nuclei from allohaploid and 59 and 43 from euploids, respectively,were photographed (captured nuclei); the remaining nuclei exhibited ba-sically the same features as those selected. A complete reconstruction ofallohaploid captured nuclei was not possible because the axial/lateral el-ements were partially destroyed in the asynaptic regions.

Statistical Analysis

Student’s t test and x2 analyses were performed using the PROC FREQand PROC TTEST procedure of SAS, respectively (SAS Institute Inc.,1999).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBLdatabases under the following accession numbers: KBr036M17,AC189325; KBrB052L10, AC189386; and KbrH117M18, AC146875.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Male Meiotic Products in Brassica napusEuploids.

Supplemental Figure 2. Male Meiotic Products in Brassica napusAllohaploids.

Supplemental Figure 3.Meiotic Spindle Orientation at Metaphase II inBrassica napus Allohaploids.

Supplemental Figure 4. Sister Chromatid Cohesion and MeioticChromosome Axes in Brassica napus Euploids.

Supplemental Figure 5. Sister Chromatid Cohesion and MeioticChromosome Axes in Brassica napus Allohaploids.

Supplemental Figure 6. Chromosome Shape and ASY1 StainingAllow Staging of Prophase I Pollen Mother Cells in Allohaploids.

Supplemental Figure 7. Electron Micrographs of Silver-StainedPachytene Nuclei in Brassica napus Euploids.

Supplemental Figure 8. Synaptonemal Complex Formation in Bras-sica napus Allohaploids Pollen Mother Cells at Pachytene.

Supplemental Figure 9. Progression of Class I Crossover Formationin Yudal Euploids.

Supplemental Figure 10. Immunolocalization of HEI10 and ASY1 inDarmor-bzh Euploids in Diplotene Pollen Mother Cells Showinga Multitude of HEI10 Foci along Chromosome Axes.

Supplemental Figure 11. Progression of Class I Crossover Formationin Yudal Allohaploids.

Supplemental Figure 12. Distribution of the Number of MLH1 Foci perCell in Euploid and Allohaploid Brassica napus.

Meiotic Crossovers in Brassica napus 1461

Supplemental Figure 13. BAC FISH Survey in Brassica napusAllohaploids: Meiotic Crossover Formation between HomoeologousChromosome Pairs A10/C9, A3/C3, and A7/C6, Respectively.

Supplemental Table 1. Distribution of MLH1/HEI10 Foci per Bivalentin Brassica napus Euploids.

ACKNOWLEDGMENTS

We thank Fabien Nogué, Christine Mézard, and Raphaël Mercier forcritical reading and discussion of the article. We also thank the twoanonymous reviewers for their constructive comments, which helped usto improve the article. Leigh Gebbie is acknowledged for Englishcorrections and I. Bancroft for providing the BAC cultures. L.G. wasfunded by a PhD fellowship from the French “Ministere de l’Enseigne-ment Superieur et de la Recherche.” N.C. was partially supported by theMinisterio 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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Meiotic Crossovers in Brassica napus 1463

DOI 10.1105/tpc.114.122788; originally published online April 15, 2014; 2014;26;1448-1463Plant Cell

Chèvre, Mathilde Grelon, Liudmila Chelysheva and Eric JenczewskiLaurie Grandont, Nieves Cuñado, Olivier Coriton, Virgine Huteau, Frédérique Eber, Anne Marie

Ploidy Does Matter!:Brassica napusHomoeologous Chromosome Sorting and Progression of Meiotic Recombination in

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