Copyright � 2008 by the Genetics Society of AmericaDOI: 10.1534/genetics.108.095893

A and C Genome Distinction and Chromosome Identification in Brassicanapus by Sequential Fluorescence in Situ Hybridization and

Genomic in Situ Hybridization

Elaine C. Howell,* Michael J. Kearsey,* Gareth H. Jones,* Graham J. King† andSusan J. Armstrong*,1

*School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom and †Rothamsted Research, Harpenden,Hertfordshire AL5 2JQ, United Kingdom

Manuscript received September 9, 2008Accepted for publication October 3, 2008

ABSTRACT

The two genomes (A and C) of the allopolyploid Brassica napus have been clearly distinguished usinggenomic in situ hybridization (GISH) despite the fact that the two extant diploids, B. rapa (A, n¼ 10) and B.oleracea (C, n¼ 9), representing the progenitor genomes, are closely related. Using DNA from B. oleracea asthe probe, with B. rapa DNA and the intergenic spacer of the B. oleracea 45S rDNA as the block, hybridizationoccurred on 9 of the 19 chromosome pairs along the majority of their length. The pattern of hybridizationconfirms that the two genomes have remained distinct in B. napus line DH12075, with no significant genomehomogenization and no large-scale translocations between the genomes. Fluorescence in situ hybridization(FISH)—with 45S rDNA and a BAC that hybridizes to the pericentromeric heterochromatin of severalchromosomes—followed by GISH allowed identification of six chromosomes and also three chromosomegroups. Our procedure was used on the B. napus cultivar Westar, which has an interstitial reciprocaltranslocation. Two translocated segments were detected in pollen mother cells at the pachytene stage ofmeiosis. Using B. oleracea chromosome-specific BACs as FISH probes followed by GISH, the chromosomesinvolved were confirmed to be A7 and C6.

IT is estimated that polyploidy has occurred in up to70% of angiosperm species and it is widely accepted

to have been a major force in the evolution of plantgenomes (reviewed by Wendel 2000). Many agriculturalcrops are polyploids, including Brassica napus (oilseedrape, Canola, swede).

The relationship between six agriculturally importantBrassica species, three diploid and three allopolyploid,was demonstrated in a classical cytogenetic study by U(1935); this relationship is commonly referred to as the‘‘U triangle.’’ That study showed that B. oleracea (genomeCC, 2n ¼ 18) has produced allopolyploids both with B.rapa (AA, 2n¼ 20) to form B. napus (AACC, 2n¼ 38) andwith B. nigra (BB, 2n ¼ 16) to form B. carinata (BBCC,2n ¼ 34). The third amphidiploid, B. juncea (AABB,2n¼36), was formed from B. rapa and B. nigra. Compar-isons among the genomes of B. rapa, B. oleracea, syntheticB. napus, and natural B. napus through molecularmarker analyses of various crosses concluded that theA and C genomes of natural B. napus have remainedessentially unaltered since the formation of the speciesand that they are similar to those of modern-day B. rapa

and B. oleracea (Parkin and Lydiate 1997; Udall et al.2005). However, it is known that B. napus cultivars differfrom each other with regard to the presence of homeol-ogous reciprocal or nonreciprocal translocations causedby recombination between homeologous regions of theA and C genomes (Parkin et al. 1995; Sharpe et al. 1995;Osborn et al. 2003; Udall et al. 2005).

Research into the genome constitution of allopoly-ploids, the pairing of homeologous chromosomes, andthe identification of translocations between chromo-somes of different genomes has been aided by recentadvances in molecular cytogenetics, particularly fluores-cence in situ hybridization (FISH) and a related tech-nique, genomic in situ hybridization (GISH) (reviewedby Raina and Rani 2001). With GISH, the total genomicDNA from one parental species is labeled and used as aprobe. Usually, an excess of unlabeled DNA (blockingDNA) from the other parent is included to increasespecificity. Because sequences common to both speciesare blocked, labeled DNA will hybridize only to genome-specific sequences on the chromosomes of its owngenome.

Identification of the diploid genomes in the allopoly-ploid brassicas, using GISH, has been successful forthose containing the B genome. Snowdon et al. (1997)demonstrated that in B. juncea and B. carinata the

1Corresponding author: School of Biosciences, University of Birmingham,Edgbaston, Birmingham B15 2TT, United Kingdom.E-mail: s.j.armstrong@bham.ac.uk

Genetics 180: 1849–1857 (December 2008)

diploid genomes could be distinguished. Maluszynska

and Hasterok (2005) combined a GISH analysis of B.juncea with FISH using labeled 5S rDNA and 45S rDNAprobes to discriminate several chromosomes withineach genome. GISH was also used in the analysis of thebehavior of B genome chromosomes in the trigenomichybrids ABC (Ge and Li 2007). However, an attempt toclearly discriminate the A genome from the C genome inB. napus by GISH was unsuccessful (Snowdon et al.1997).

We present a strategy to distinguish between the Aand C genomes in B. napus and to identify individualchromosomes within a genome. It is based on acytological technique using material from anthers,which provide many meiotic cells as well as mitotic cellsfrom the tapetal layer. We demonstrate that ourmodified GISH technique, which includes a repetitiveprobe in the block, is effective on both mitotic andmeiotic cells. We have used it predominantly on meioticpachytene chromosomes and on chromosomes at latediakinesis. Further, we have developed a sequentialprocedure with FISH and GISH that allows us to identifyseveral specific chromosomes. Using a B. napus cultivarcarrying a known reciprocal translocation, we alsodemonstrate that it is possible to visualize intergenomictranslocations and to identify the chromosomes in-volved by the sequential use of FISH with BACs fromB. oleracea, which are linked to a genetic map (Howell

et al. 2005) and GISH.

MATERIALS AND METHODS

Slide preparation: Anthers with pollen mother cells atmeiotic stages between prophase I and metaphase I werecollected from B. napus doubled haploid line DH12075(provided by D. Lydiate, AAFC, Saskatoon, Saskatchewan,Canada) and from B. napus cv. Westar and N-o-1, a DH linederived from Westar (provided by John Innes Centre, Nor-wich, UK). Methods for staging and fixing anthers have beendescribed previously (Armstrong et al. 1998; Howell et al.2002). Slides were prepared from these anthers followingenzyme digestion (Armstrong et al. 1998; Howell et al. 2002)but cytohelicase was omitted from the enzyme mixture and thedigestion time was extended to 2 hr. Slides were screened forwell-spread chromosomes that were free of cytoplasm.

Probes and blocking DNA: For GISH, total genomic DNAwas isolated from young leaf tissue of B. oleracea var. alboglabradoubled haploid line A12DHd (C genome) and B. rapacultivar Golden Ball (A genome) using a DNA extraction kit(Tepnel Life Sciences). After treatment with RNase andquantification against l-phage DNA standards (Sigma) bygel electrophoresis, some of the DNA was mechanicallysheared and then labeled with biotin-16-dUTP by nick trans-lation (Roche). The remainder was autoclaved for 5 min twiceto obtain DNA fragments of 100–500 bp for use as blockingDNA. The intergenic spacer (IGS) region of B. oleracea 45SrDNA was amplified from A12DHd genomic DNA by PCRusing primers based on EMBL X60324. The primerswere Fwd:CAGCCCTTTGTCGCTAAG (within 25S gene) andRev:GGCAGGATCAACCAGGTA (within 18S gene) and theannealing temperature was 61� (Howell et al. 2002). The PCR

product was cleaned and concentrated through a PCRpurification kit (Qiagen) and used for blocking.

The probes used for FISH were (1) 45S rDNA from clonepTa71 (Gerlach and Bedbrook 1979), EMBL X07841,labeled with digoxigenin-11-dUTP; (2) BoB061G14, a BACclone that hybridizes to the pericentromeric heterochromatinof six B. oleracea chromosomes, labeled with biotin-16-dUTP;and (3) BoB028L01, BoB057M06, and BoB028I05, three BACclones assigned to B. oleracea chromosome C6 (BolC6)(Howell et al. 2002, 2005). The first two BACs were labeledwith digoxigenin-11-dUTP and the third was labeled withbiotin-16-dUTP. All probes were labeled by nick translation(Roche).

In situ hybridization: Slide pretreatment, chromosome andprobe denaturation, and hybridization and post-hybridizationtreatments were the same for GISH and FISH and followedprevious methods (Howell et al. 2002) except that denatur-ation at 75� was reduced to 3 min 30 sec. Probes were appliedto a slide in 20 ml of a probe mixture that contained 50%deionized formamide, 23 SSC, 10% dextran sulfate, labeledprobes, and blocking DNA if required.

For GISH, 50–100 ng labeled C genome DNA and �500 ngor 1100 ng blocking A genome DNA were used, with or without100 ng IGS DNA. On two slides, labeled A genome DNA(50 ng) was used with 5000 ng blocking C genome DNA.

For FISH,�20–50 ng of each labeled probe was included inthe probe mixture. Either both 45S rDNA and BoB061G14were used or the three chromosome BolC6 BACs were usedsimultaneously. Approximately 1 mg C0t -1 DNA, preparedfrom A12DHd, was added with the BolC6 BACs to blockrepetitive sequences.

Biotin-labeled DNA and dioxigenin-labeled DNA were de-tected with Cy3 streptavidin (Cambio) and antidigoxigenin-fluorescein (Roche), respectively. Slides were counterstainedwith 49,6-diamidino-2-phenylindole (DAPI; 1 mg/ml) in Vecta-shield (Vector).

After image capture, FISH slides selected for sequentialprobing with GISH were soaked in 23 SSC for 10 min toremove the coverslip, dehydrated through an ethanol series(70 and 85% for 30 sec and 100% for 1 min) and air dried. AGISH probe mixture was applied and the chromosomes andprobes were denatured together at 75� for 3 min. Theprocedures following this step were the same as those for thefirst probing.

Image capture: Images were captured and analyzed usingSmartCapture 2 software (Digital Scientific UK) and a Photo-metrics Sensys CCD camera attached to a Nikon E600fluorescence microscope. When slides were probed sequen-tially, recapture of the same chromosome spreads was facili-tated by an automated stage. In all images, the hybridizationsites of Cy 3 streptavidin-detected probes are red, the sites ofantidigoxigenin–fluorescein-detected probes are green, andDAPI staining is blue.

RESULTS

GISH: When labeled total genomic DNA from B.oleracea (C genome) was hybridized to mitotic or meioticchromosomes from anthers of B. napus line DH 12075 inthe presence of blocking DNA from B. rapa (A genome),nine pairs of chromosomes were strongly labeled. This isexpected if the chromosomes of B. oleracea (n ¼ 9) arehomologous to the C genome chromosomes of B. napus(n ¼ 19). With the higher concentration of blockingDNA (1100 ng/slide) the differentiation between the

1850 E. C. Howell et al.

genomes was clear. With the lower concentration ofblocking DNA (500 ng/slide), the nine C chromosomepairs could still be distinguished but the differentiationwas reduced due to increased labeling of the pericentro-meric regions of the other chromosomes (not shown).Repeatability was confirmed using DNA samples that hadbeen extracted, labeled, and autoclaved on a differentoccasion.

Even with the higher concentration of blocking DNA,the signal strength was not uniform along the chromo-somes. In general, the intensity decreased from thepericentromeric regions toward the telomeres but verystrong signals occurred at one end of each chromosomeof two labeled pairs. It was likely that these signals werefrom C genome 45S rDNA sites and therefore anunlabeled PCR product of the IGS region of B. oleracea45S rDNA was added to the probe mixture. This resultedin a reduction in the intensity of the strongest signalsand therefore produced a more even distribution of thehybridization signal intensity in both mitotic (Figure1A) and meiotic (Figure 1, B and C) preparations. Onpachytene spreads, the coalesced centromeric regions(synizetic knot) fluoresced brightly and the hybridiza-tion signals became fainter and less frequent toward thetelomeres, with the telomeric and subtelomeric regionsbeing unlabeled. Nevertheless, with this combination ofprobe, IGS, and A genome block, the free chromosomearms could be categorized as labeled or unlabeled (notshown).

In contrast, when A genome DNA was used as the labeledprobe with C genome DNA as the block (ratio of 1 probeto 100 block) on preparations at metaphase I, hybrid-ization was restricted to limited regions on 10 bivalents.

The signals differed in strength and the bivalents withthe weakest signals were not clearly differentiated fromsome C genome bivalents, which fluoresced slightly atpericentromeric regions (not shown). This combina-tion was not investigated further.

FISH with repeat probes followed by GISH: We thenused FISH (Figure 1D) with 45S rDNA and BoB061G14,which hybridizes to pericentromeric regions of manychromosomes, followed by GISH (Figure 1E) on prep-arations of DH12075 at diakinesis. The distribution ofthe signals from these probes among the chromosomesof the A and C genomes is shown in Table 1. The signalsdiffered in intensity; for example, the strongest 45SrDNA signal was located distally on an A genomebivalent that lacked BoB061G14 signals and the weak-est BoB061G14 signal was on an A genome bivalent.Although the chromosome preparations deterioratedslightly with this sequential probing, the two genomeswere clearly differentiated. Without the addition ofunlabeled IGS DNA, strong GISH signals were seen atthe 45S rDNA sites on two C genome bivalents (Figure 1,D and E). In contrast, none of the A genome 45S rDNAsites showed labeling with GISH. Associations between45S rDNA sites were seen frequently within and betweengenomes at diakinesis.

GISH on Westar: Genetic linkage mapping with RFLPsestablished that a reciprocal translocation involving thelower portions of linkage groups A7 and C6 is present inseveral annual oilseed B. napus genotypes, including thecultivar Westar (Osborn et al. 2003). The translocationappears to be interstitial on the basis of segregation datafor one or two markers at the end of these linkage groupsin different populations and the presence of two synapsis

Figure 1.—GISH and sequentialFISH and GISH on preparations of B.napus at (A) mitosis and (B–F) meioticdiakinesis. GISH with C genome DNAas probe (red) and A genome DNA asblock. (A–E) DH12075; (A and B)GISH, with IGS included in the block;(C) image B with red filter only; (D)FISH with 45s rDNA (green) andBoB061G14 (red); (E) reprobe of prep-aration (D) by GISH without IGS in theblock; (F) Westar, GISH showing a C ge-nome segment on an A genome bivalent(arrow). Chromosomes counterstainedwith DAPI. Bar: A, 5 mm; B (for B–F),5 mm.

FISH and GISH on B. napus 1851

exchange points in synaptonemal complex spreads of anF1 hybrid between DH line N-o-1, which was derived fromWestar, and a line not carrying the translocation (Sharpe

et al. 1995; Osborn et al. 2003).When preparations of Westar undergoing mitosis

or diakinesis (Figure 1F) were subjected to GISH, weidentified nine labeled pairs, nine unlabeled pairs, andone pair with labeling confined to one end. Weconclude that one A genome pair had a translocationfrom a C genome pair. Although a reciprocal trans-location could not be identified with certainty at thesestages, on good pachytene spreads, where long lengthsof bivalents were free from the synizetic knot, onepredominantly unlabeled bivalent had a long, labeled,distal section and, conversely, a labeled bivalent had anunlabeled section of similar size at the end (not shown).This unlabeled section showed no hybridization signalsclose to the telomere. Hybridization signals on thetranslocated labeled section decreased toward thetelomere, with the telomeric and subtelomeric regionsbeing unlabeled, similar to other labeled bivalents.Although our technique was sensitive enough to detectthese translocations, we wanted to confirm that theywere the result of a reciprocal translocation and thatthey represented the reciprocal translocation identifiedpreviously with molecular markers.

FISH with B. oleracea BACs followed by GISH onDH12075, Westar, and DH line N-o-1: First we used threeB. oleracea BACs, previously assigned near the bottom ofchromosome BolC6 (Howell et al. 2005), as FISH probesto determine whether we could identify the correspondingregion in the C genome of B. napus. Second, we inves-tigated whether we could establish that the translocationsvisualized by GISH in Westar involved the regions iden-tified by linkage mapping, since BolC6 corresponds tolinkage group B. napus C6 (BnaC6) (Parkin et al. 1995).

The BAC probes, tested in pairs on pachytene spreadsof DH12075, hybridized to two bivalents in positionssimilar to those on chromosome BolC6 (Howell et al.2005), with BoB028I05 (red) nearest the telomere,BoB028L01 (green) nearby, and BoB057M06 (green)

farther away from the telomere. Signals were oftenstronger on one bivalent. Subsequently, FISH, with allthree BACs applied simultaneously (Figure 2A), wasfollowed by GISH (Figure 2C). One hybridization site ofBoB057M06 (green) is obscured in Figure 2A but wasseen in other images. The bivalent with the strongestBAC signals was labeled by GISH whereas the secondbivalent was not. This indicates that the BACs preferen-tially hybridized with the B. napus C genome and thatthere was sufficient sequence homology between theBACs and the homeologous region in the A genome forsome hybridization to occur there as well.

On Westar, the three BACs gave the same pattern ofhybridization signals as on DH12075 (Figure 2B).However, on reprobing with GISH and comparing thesequential images, a distinct change from labeled tounlabeled chromatin could be seen between the twogreen signals (Figure 2, B, D, and F). Therefore, on onebivalent, the BoB028I05 and BoB028L01 sites were on aGISH-labeled section while the BoB057M06 site was onan unlabeled section whereas on the other bivalent thereverse situation occurred. The sequential probing ofDH12075 and Westar is shown diagrammatically inFigure 2, E and F, respectively. This confirmed that thetranslocation observed in Westar is reciprocal andinvolves regions of A and C genome chromosomes thathave homology to part of BolC6.

DH line N-o-1, probed sequentially with the BACs andGISH, gave results that were indistinguishable fromthose of Westar. The images of whole bivalents (Figure 3,A–H) illustrate the proportion of the chromosomesinvolved in this reciprocal translocation. Although everyBAC hybridization signal is not present on these images,with BoB057M06 being the least reliable, the sites wereobserved on other pachytene spreads. The occasionalpresence of a faint green site near the BoB028L01 site(Figure 3, C and D) suggests that this BAC hybridizesweakly to a duplicate region that was identified in BolC6(Howell et al. 2005) and likely to be present in BnaC6.Some cross-hybridization to the A genome pericentro-meric heterochromatin can be seen. The translocated

TABLE 1

The distribution of FISH signals of 45S rDNA and BAC BoB061G14 on the A and C genomechromosomes of B. napus

No. of chromosome pairs

FISH signals Full complement (19) A genome (10) C genome (9) Chromosomes

45S only 3 2 1 A3 A5C8

BoB061G14 only 10 5 5 A2 A4 A7 A8 A10C1 C2 C4 C5 C6

45S 1 BoB061G14 4 3 1 A1 A6 A9C7

None 2 0 2 C3 C9

1852 E. C. Howell et al.

section of a C genome chromosome on the A genomepair seen at diakinesis could also be identified as part ofBnaC6 by using these BACs prior to GISH on spreads atdiakinesis (Figure 3, I and J).

DISCUSSION

The diploid species B. rapa (A genome), B. nigra (Bgenome), and B. oleracea (C genome), which representthe progenitors of the three allotetraploids of U’striangle (U 1935), are closely related. Molecular phylo-genetic analyses of the tribe Brassiceae within the familyBrassicaceae place B. nigra in a different lineage toB. oleracea and B. rapa (Warwick and Black 1991).These lineages split �7.9 million years ago (MYA)(Lysak et al. 2005) whereas B. oleracea and B. rapadiverged more recently, �3.75 MYA (Inaba and Nishio

2002). This close relationship is also indicated fromFISH experiments because only a few probes appear tobe species specific. Some sequences used as probes,such as the ‘‘centromeric retrotransposon of Brassica’’and a tandem repeat (TR805), hybridized to chromo-somes of all three species (Lim et al. 2007) whereasothers, for example, the tandem repeat sequencespBcKB4 (CentBr1), pBoKB1, and CentBr2, hybridizedto several chromosomes of both the A and C genomes

but not to the B genome (Harrison and Heslop-Harrison 1995; Lim et al. 2005, 2007).

It has been suggested that this similarity would pre-vent the successful application of GISH to B. napus.Excessive cross-hybridization in pericentromeric regionsand a low level of hybridization in chromosome armswas observed in one GISH experiment regardless ofwhich genome was used as the labeled probe (Snowdon

et al. 1997). We have shown that the A and C genomescan be distinguished clearly, provided that B. oleracea(C genome) DNA is used as the probe and B. rapa (Agenome) DNA as a block, with the addition of the Cgenome IGS sequence to the block improving thedifferentiation.

When we used labeled A genome DNA with C genomeDNA as a block, the pattern of labeling suggested thatA-genome-specific sequences are concentrated in thepericentromeric regions and rDNA arrays. ‘‘Pericentro-meric retrotransposon of Brassica rapa’’ (a group of fourfamilies of Ty3-Gypsy elements) and a degeneratetandem repeat (TR238) are likely to be among thesesequences because they hybridized to pericentromericheterochromatin of several A genome but no C genomechromosomes in a FISH experiment (Lim et al. 2007).

In contrast, we conclude that C-genome-specific se-quences must be widely distributed across the chromo-somes because the GISH labeling was widespread when

Figure 2.—FISH and sequen-tial GISH identify a reciprocaltranslocation in Westar. (A andB) FISH with three BolC6 BACs(red and green) on partial pachy-tene spreads of (A) DH12075 and(B) Westar. White arrows indicatebivalents with BAC hybridizationsites. (C and D) Reprobing of Aand B with GISH (C genomered), including IGS in the block;white arrows indicate the samechromosomes as in A and B; out-lined arrows in D indicate translo-cation breakpoints. (E and F)Diagrams amalgamating A withC and B with D, respectively, toshow homeologous regions ofC6 and A7 in DH12075 and thetranslocated segments in Westar.Pink line, C genome C6; blue line,A genome A7; dotted line, expectedcontinuation of bivalent—arrowat end of line points toward cen-tromere. Red circle 1, BoB028I05;green circle 2, BoB028L01; greencircle 3, BoB057M06. Bar, 5 mm.

FISH and GISH on B. napus 1853

the C genome was the probe. It is likely that severaldifferent types of sequence are involved and, becauseevery C-genome-specific sequence is labeled, the cover-age that we obtain with GISH is better than that achiev-able with an individual transposable element or repetitivesequence.

A telomere-like repetitive sequence, pBo1.6, ap-peared to be C genome specific with FISH. It waslocalized at interstitial and/or telomeric/subtelomericregions of all B. oleracea chromosomes and detected on18–24 chromosomes in B. napus. However, although itwas rarely detected on B. rapa chromosomes by FISH, ithybridized to B. rapa DNA on a Southern blot (Galvao

Bezerra Dos Santos et al. 2007). In our GISH experi-ments, we used a high concentration of A genome DNAin the block and it is likely that there were enoughcopies of A genome pBo1.6 to hybridize with the moreabundant C genome sites. As the C genome telomericand subtelomeric regions were not labeled in our GISHexperiments, we suggest that the pBo1.6 sites wereblocked. The minimum level of sequence similarityrequired for a sequence from the A genome to effectivelyblock a C genome site is not known but is proba-bly .85% because, despite there being 82–85% se-quence similarity between the CentBr1 and CentBr2repeat sequences, a CentBr1 probe did not hybridize toCentBr2 sites in B. rapa FISH experiments (Lim et al.2005).

Sequences within a BAC, BoB014O06, from B. oleraceamay be among the GISH C-genome-specific sequences.This BAC specifically hybridized to all C genomechromosomes in B. napus in FISH experiments, al-though the hybridization signals were not homoge-neous along the chromosomes (Leflon et al. 2006;Nicolas et al. 2007). One component of this BAC isBot1, a recently identified CACTA transposon, which ishighly amplified in the C genome with an estimated1500 copies in the B. oleracea haploid genome and only13–177 in B. rapa. When one copy was used as a FISHprobe on B. napus, all the C genome chromosomes werelabeled whereas only minor hybridization signals weredetected on A genome chromosomes. A 2.7-kb region ofthe Bot1 sequence was identified as C genome specific(Alix et al. 2008). Therefore, although some regions ofthe Bot1 sequence may be blocked by A genome copies,this 2.7-kb region is likely to be involved in the GISHlabeling of the C genome.

Other transposable elements are likely to be amongthe C-genome-specific sequences. Approximately 20%(120 Mb) of the B. oleracea genome is composed oftransposable elements with class 1 elements, includingboth LTR and non-LTR retrotransposons, accountingfor 14%. The remaining 6% is composed of class 2(DNA) elements with the most abundant superfamilybeing CACTA (Zhang and Wessler 2004). Manysubfamilies of retroelements are present in both the A

Figure 3.—FISH and se-quential GISH showingthe reciprocal translocationin DH line N-o-1, derivedfrom Westar. (A–D) FISHwith three BolC6 BACs(red and green) on biva-lents at late pachytene.Some hybridization siteswere not detected on theseparticular spreads; thosedetected are shown bywhite arrows. (E–H) Rep-robes of A–D with GISH(C genome red) with IGSin the block. (A, E, B, andF) C6 bivalent with a trans-located segment of A7. (C,G, D, and H) A7 bivalentwith a translocated segmentof C6. Outline arrow showsposition of translocationbreakpoint determined byGISH. (I and J) A7 withC6 translocation at diakine-sis identified with (I) FISHand ( J) sequential GISH.Bar, 5 mm.

1854 E. C. Howell et al.

and C genomes but a few that may be C genome specifichave been identified through sequence analysis of PCRproducts from retroelements in the species of U’striangle (Alix and Heslop-Harrison 2004).

We have established that the IGS of the 45S (18S-5.8S-25S) rDNA genes is a component of the C-genome-specific sequences in our GISH experiments. Thecoding sequences of the 45S and 5S rDNA genes arehighly conserved in plants and, consequently, those inthe C genome will be blocked. In brassicas, while somesequence variation occurs in the internal transcribedspacer within the set of 45S rDNA genes (Yang et al.1999), the IGS between the tandemly arrayed sets ismuch more divergent (Bhatia et al. 1996). In ourexperiments, the A genome DNA blocked the 45S rDNAsites of the A genome but not those of the C genome.Addition of unlabeled B. oleracea IGS to the blockresulted in reduced hybridization signals from thesesites, indicating that the IGS in the B. rapa genomicDNA had not hybridized effectively to them. The IGS ofB. oleracea A12DHd may not be completely homologousto the IGS of the B. napus C genome since the sites werestill not totally blocked. However, total blocking was notrequired because we wanted to label as much of the Cgenome chromosomes as possible.

The GISH labeling of the C genome chromosomesindicates that extensive intergenomic sequence homog-enization has not occurred in B. napus. Homogeniza-tion of the rDNA arrays has occurred in some polyploids(reviewed by Wendel 2000) and it is occurring rapidlyin the rDNA of two Tragopogon allotetraploids, whichoriginated within the last 80 years (Kovarik et al. 2005).Although we saw frequent associations between 45SrDNA sites within and between genomes at diakinesis,these were probably remnants of nucleolar associationsfrom the preceding interphase (Hasterok et al. 2005)and it is unlikely that recombination regularly occurs inthese regions. The fact that the IGS of the 45S rDNAarrays is genome specific in our GISH experimentsindicates that the two parental 45S rDNA array typeshave been maintained in B. napus. This is supported byevidence from blots of restriction-enzyme-digested DNAprobed with an rDNA repeat (Waters and Schaal

1996) and from comparisons of the promoter sequen-ces in the IGS (Chen and Pikaard 1997). This impliesthat recombination between the 45S rDNA arrays of thetwo genomes is rare and/or that a mechanism such asgene conversion is acting within but not betweengenomes.

The absence of large-scale translocations betweenthe genomes in DH12075 is evident from the fact thatC genome chromosomes were labeled along almosttheir entire length apart from the telomeres and asubtelomeric region. The only labeling of A genomechromosomes occurred faintly in pericentromeric het-erochromatin, most probably due to cross-hybridizationrather than translocation. This supports the conclusion

drawn from RFLP mapping data that the distinct A andC genomes have been maintained to a large extent innatural B. napus throughout its evolution despite thefact that homeologous recombination occurs both inmodern natural cultivars (Sharpe et al. 1995; Udall

et al. 2005) and in resynthesized B. napus lines (Parkin

et al. 1995; Udall et al. 2005). Individual natural B.napus cultivars possess small numbers of homeologousreciprocal or nonreciprocal translocations (Parkin et al.1995; Sharpe et al. 1995; Osborn et al. 2003; Udall et al.2005) but over the course of the evolution of this speciesthere has not been a significant mixing or homogeni-zation of the genomes, which suggests that most non-reciprocal translocations cause a reduction in fitnessand that selection has acted against them.

Using GISH we cannot see if rearrangements haveoccurred within a genome in B. napus compared to thediploids, but no substantial rearrangements were iden-tified when genetic maps were compared (Parkin et al.1995; Bohuon et al. 1996; Parkin and Lydiate 1997).Therefore, it is likely that the A and C genomechromosomes in B. napus are similar to the chromo-somes in the diploids and we can compare them usingFISH and GISH. In a study of several species of theBrassicaceae, inter- and/or intravarietal polymorphismin the distribution of rDNA sites was observed (Hasterok

et al. 2006). However, comparisons between the A and Cdiploid species and B. napus (Snowdon et al. 2002) orB. napus haploid lines (Kamisugi et al. 1998) have shownconservation of the number and location of 45S rDNAsites and only minor differences in signal strength.Therefore, the location but not the signal strength of45S rDNA loci in the diploid genomes may be used toidentify homologs in B. napus with some confidence.With 45S rDNA and BoB061G14 as FISH probesfollowed by GISH, we were able to distinguish severalchromosomes of B. napus and to identify potentialhomologs in the karyotypes of B. oleracea (Howell

et al. 2002) and B. rapa (Lim et al. 2005). Since thechromosomes of the diploids have been associated withlinkage groups of genetic maps (Howell et al. 2002;http://www.brassica.info) and molecular marker analy-sis has confirmed homology between these linkagegroups and those of B. napus (A1-10 ¼ R1-10 ¼ N1-10and C1-9 ¼ O1-9 ¼ N11-19) (Parkin and Lydiate

1997), we can tentatively assign linkage groups to thesechromosomes. Subsequently, five of these have beenconfirmed using B. oleracea chromosome-specific FISHprobes.

The C genome chromosomes of B. napus lineDH12075 showed the same distribution of signals asthose of B. oleracea A12DHd except that the interstitial45S rDNA signal of BolC4 was not present (Table 1).Since this site is absent from other B. oleracea accessions,this was expected. C7 has a BoB061G14 signal and adistal 45S rDNA signal. C8 has a distal 45S rDNA signalbut the signal intensity is less than in A12DHd. Neither

FISH and GISH on B. napus 1855

probe hybridizes to C3 and C9 (confirmed, not shown)and these chromosomes can be differentiated by sizeand centromere position in mitotic spreads (not shown).The remaining five chromosomes form a group withonly BoB061G14 signals. The pattern of hybridization of45S rDNA and BoB061G14 in the A genome was thesame as that described for 45S rDNA and CentBr1 in B.rapa (Lim et al. 2005). Therefore, we can identify two Achromosomes and divide the remainder into two groups(Table 1). A3 has a large distal 45S rDNA signal(confirmed, not shown) and A5 has a 45S rDNA signalnear the centromere. A1, A6, and A9 have both 45SrDNA and BAC signals and the remaining five have onlya BAC signal. We have also shown that B. oleracea BACscan effectively mark chromosome BnaC6 and, to a lesserextent, the homeologous part of BnaA7. It should bepossible to mark any particular homolog in B. napus withBACs from the diploids because chromosome-specificBACs have been identified for all of their chromosomes(http://www.brassica.info).

Many B. napus cultivars studied with molecularmarkers have been shown to have homeologous re-ciprocal or nonreciprocal translocations (Parkin et al.1995; Sharpe et al. 1995; Osborn et al. 2003; Udall et al.2005). The one that has been investigated most thor-oughly is an interstitial reciprocal translocation involv-ing A7 and C6, which is present in several Canadian andAustralian cultivars. The source of this translocationmay be one of three annual forms of B. napus, which arecommon to all their pedigrees (Osborn et al. 2003).With sequential FISH and GISH, on pachytene spreadswe identified this reciprocal translocation in Westar andN-o-1. The proximal end of the translocation wasbetween the signals from BoB057M06 and BoB028L01and therefore between markers pO10E1 and BoAP1-c(Howell et al. 2005), agreeing with the genetic markerdata that places it below pO10eNP (Osborn et al. 2003).However, we were unable to confirm that the trans-location was interstitial as there was no sign of GISHlabeling near the telomeric end of the unlabeledsegment. This suggests that the distal breakpoint wasin the small region close to the telomere where labelingin the C genome was absent. The fact that the moredistal synapsis exchange point of the two indicated in asynaptonemal complex spread of an F1 hybrid N-o-9 3

N-o-1 (Osborn et al. 2003) was not far from the telomeresupports this suggestion. The exchanged region wasapproximately one-third of the length of the A7 andC6 chromosomes in synaptonemal complex spreads(Osborn et al. 2003) and our images of the wholechromosomes at pachytene agree with this. C6 is thesmallest chromosome of the C genome complement(Howell et al. 2002).

At diakinesis, the A7 chromosome with the segmentfrom the C genome can be seen clearly but the C6chromosome with the A genome segment cannot and itis also not easy to identify this chromosome in every

spread at mitosis. Using GISH on mitotic spreads ofwheat containing recombined wheat–rye chromo-somes, a small labeled translocated segment was de-tected on an unlabeled chromosome. However, whenthe same segment was not labeled but the chromosomewas labeled, the segment could not be detected and itwas suggested that the halo effect of the probe obscuredthe unlabeled segment (Lukaszewski et al. 2005). Thisimplies that, since we can label only the C genome inB. napus, small translocations of C genome chromo-somes will be easier to detect than the reciprocaltranslocations. As noted above, small translocationsinvolving only the unlabeled subtelomeric regions willnot be detected by GISH.

We have found that using FISH to assign BACscontaining paralogous genes to their respective linkagegroups is a useful alternative to genetic mapping inB. oleracea, particularly if polymorphism is difficult todetect. Using pachytene spreads of B. oleracea, we canalso assist the sequencing of some regions by confirmingthe order and relative position of selected BACs(Howell et al. 2005). Now that we have developedGISH and shown that B. oleracea BACs can be used onB. napus to identify chromosomes, it will be possible toallocate B. napus BACs to linkage groups in a similar way.

We are using both GISH and FISH on pollen mothercells at metaphase I to study the genetic control ofhomeologous pairing and recombination in B. napus.Experiments with B. napus haploids (AC) showed thatthe amount of pairing between chromosomes varieddepending on the varieties from which the haploidsoriginated and that this was controlled by a major locus,PrBn, together with other loci. However, the B. napusvarieties themselves (AACC) had normal bivalent pair-ing ( Jenczewski et al. 2003; Liu et al. 2006). Fromgenetic mapping, it is apparent that the frequency ofhomeologous recombination is much higher in resyn-thesized than in natural B. napus (Parkin et al. 1995)and it may be possible to exploit this situation todetermine whether different loci or different alleles atthe PrBn locus are involved in the control of homeolo-gous recombination in B. napus. The techniques willalso be useful in evolutionary studies on newly synthe-sized B. napus allopolyploids because some of theintergenomic translocations resulting from homeolo-gous recombination can be identified.

We are grateful to S. Price and K. Staples (University of Birmingham,Birmingham, UK) for technical assistance and to I. A. Parkin (AAFC,Saskatoon, Saskatchewan, Canada) and C. Morgan ( JIC, Norwich,UK) for seed. This work was supported by the Biotechnology andBiological Sciences Research Council (grant no. G18621).

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Communicating editor: A. H. Paterson

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