The genomic organization of retrotransposons in Brassica oleracea

Karine Alix1,2, Carol D. Ryder3, Jay Moore3, Graham J. King4 and J.S.(Pat)Heslop-Harrison1,*1Department of BiologyUniversity of Leicester, LE1 7RH, Leicester, UK (*author for correspondence; e-mailphh4@le.ac.uk); 2UMR de Genetique Vegetale INRA/UPS/CNRS/INA P-G, Ferme du Moulon, 91190, Gifsur Yvette, France; 3Warwick HRI, Wellesbourne, CV35 9EF, Warwick, UK; 4Rothamsted Research,Harpenden, AL5 2QJ, Hertfordshire, UK

Received 29 April 2005; accepted in revised form 1 August 2005

Key words: BAC library, copia, fluorescent in situ hybridization (FISH), gypsy, LINE, reverse transcriptase

Abstract

We have investigated the copy numbers and genomic organization of five representative reverse trans-criptase domains from retrotransposons in Brassica oleracea. Two non-homologous Pseudoviridae (Ty1/copia-like) elements, two Metaviridae (Ty3/gypsy-like) elements (one related to the Athila family) and oneRetroposinae (LINE) element were hybridized to a gridded BAC library, ‘‘BoB’’. The results indicated thatthe individual LTR retrotransposons (copia and gypsy-like) were represented by between 90 and 320 copiesin the haploid genome, with only evidence of a single location for the LINE. Sequence analysis of the sameelements against genome survey sequence gave estimates of between 60 and 570, but no LINE was found.There was minimal evidence for clustering between any of these retroelements: only half the randomlyexpected number of BACs hybridized to both LTR-retrotransposon families. Fluorescent in situ hybrid-ization showed that each of the retroelements had a characteristic genomic distribution. Our results suggestthere are preferential sites and perhaps control mechanisms for the insertion or excision of differentretrotransposon groups.

Introduction

Class I transposable elements (TEs), or retro-transposons, represent a major fraction of allplant genomes (Kumar and Bennetzen, 1999), andare important factors in generation of diversitybecause of their ability to transpose from onegenomic site to another. Retrotransposons aredivided into two groups, those that are flanked bylong terminal repeats (LTRs) including Ty1/copiaand Ty3/gypsy retrotransposons, and non-LTRretrotransposons or LINEs (long interspersednuclear elements) (Xiong and Eickbush, 1990).As retrotransposons are widespread in the plantkingdom (Flavell et al., 1998), the distribution of

copia and gypsy-like retrotransposons have beenanalyzed in various plant genomes using fluores-cent in situ hybridization (FISH) (e.g. Brandeset al., 1997, Kumar et al., 1997, Friesen et al.,2001, Mroczek and Dawe, 2003) and there isevidence that individual retroelement familieshave typical patterns of chromosomal localization(e.g. Presting et al., 1998; Belyayev et al., 2001;Friesen et al., 2001). In addition, the availabilityof the complete sequence of the Arabidopsisthaliana genome (AGI, 2000) and of large geno-mic sequences from different plant species (e.g.maize, rice, barley) has allowed the analysis of thegenomic distribution of retrotransposons, reveal-ing a non-random distribution of TEs along the

Plant Molecular Biology (2005) 59:839–851 � Springer 2005DOI 10.1007/s11103-005-1510-1

genome (Peterson-Burch et al., 2004) and notablythe existence of preferential TE-enriched regions(SanMiguel et al., 1996; Walbot and Petrov,2001). Therefore, characterization of the genomicorganization and distribution of retrotransposonsin the plant kingdom helps further our under-standing of the factors contributing to plantgenome remodelling.

Brassica is an agriculturally important dicoty-ledonous genus that provides a wide range ofdiploid and allotetraploid species (U, 1935) closelyrelated to the model plant Arabidopsis thaliana(they belong to the same taxonomic family,Cruciferae or Brassicaceae). Arabidopsis and theBrassica species are thought to have evolved viarounds of genome duplications (Lagercrantz,1998; Vision et al., 2000; Simillion et al., 2002)after divergence from a common ancestor 15–20million years ago. For instance, the diploid speciesB. oleracea has a complex genome, which isextensively triplicated (Lagercrantz and Lydiate,1996; O’Neill and Bancroft, 2000). Evidence ofsequence conservation and collinearity of geneorder in many genomic regions support thistheory (e.g. Kowalski et al., 1994; Cavell et al.,1998; Ryder et al., 2001; Babula et al., 2003).In addition to the comparative survey of thecoding regions in Arabidopsis and Brassica, com-parative analyses of the non-coding fraction ofthe genome, mainly represented by repetitivesequences and TEs, would be of particular interestto understand the global genome evolution sincedivergence from the common ancestor of theBrassicaceae.

As in all other plant species investigated, copiagroup retrotransposons can be isolated from B.oleracea and B. rapa. In situ hybridization hasshown that they are abundant and distributedalong all chromosomes with higher density in somechromosomal regions (Brandes et al., 1997). Re-cently, Zhang and Wessler (2004) have comparedthe TEs in the A. thaliana sequence (125 Mb) withthose in genomic survey sequence of B. oleracea(genome size 650 Mb, almost five times larger) toestimate the patterns of amplification, diversifica-tion, and loss since divergence of the species froma common ancestor. They found nearly all TElineages were shared by the species, concludingthat both species inherited and retained largely thesame collection of TEs from their common ances-tor, and amplification of TEs contributes largely to

the difference in genome size. Given the impor-tance of genome size changes in species evolution,we aimed to learn more about the genomicorganization (clustering and/or distribution) andmechanisms (inferred from any occurrence of non-random events) of the more abundant retroele-ment classes in Brassica.

Large insert genomic libraries offer a uniqueopportunity to study the genomic organizationand copy numbers of repetitive DNA sequencemotifs such as retrotransposons. The B. oleracea‘‘BoB’’ BAC (bacterial artificial chromosome)library, developed at Warwick-HRI, is wellcharacterized as a result of use in numerousresearch projects aimed at gene isolation, tar-geted sequencing and physical mapping of theB. oleracea genome (http://brassica.bbsrc.ac.uk/IGF/). From this work over 100 hybridizationscreening data sets and additional characteriza-tion data are available. These include screenswith repetitive, ribosomal, chloroplast and cen-tromeric sequences, RFLP marker probes andsingle/low copy functional genes. Many of theseare mapped to diverse Brassica chromosomesand are homologous to Arabidopsis sequences.BACs provide efficient hybridization probes forlocalization of sequences in situ on chromosomes(Fransz et al., 1998; Cheng et al., 2001; Howellet al., 2002) and the use of ‘‘BoB’’ BACs hasallowed the integration of the cytogenetic andgenetic linkage maps of B. oleracea (Howellet al., 2002). In barley, a screen with gypsyretrotransposons showed that the elementhybridized to about 3% of the BAC colonies(Vershinin et al., 2002), and showed little evi-dence for clustering of multiple copies of thesequences in single BACs. These results comple-ment genomic studies using sequencing, in situand Southern hybridizations or PCR, and enablethe large-scale structure of the genome to bemodelled.

In the present study, we examine the abun-dance and genomic distribution of retrotranspo-sons that were previously isolated from Brassicaspecies (Alix and Heslop-Harrison, 2004) in thediploid species B. oleracea. Results will assist inunderstanding the evolution of the species andgenomes in the genus, the organization ofretrotransposons with respect to genes, andfurther physical mapping of sequences in thegenome.

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Materials and methods

Reverse transcriptase probes

Five clones containing the RT domain fromvarious retrotransposons isolated from B. oleracea(Bo) or B. napus (Bn) were used: two copiaelements, two gypsy elements and a LINE. Thetwo copia elements used were Bo10COP-16(266 bp) and Bo10COP-18 (263 bp) (EMBL acces-sion numbers: AJ414054, AJ414055 respectively).They share only 55% sequence homology at thenucleic acid level and represent the diversity withinthe B. oleracea copia family. The two diverse gypsyelements used were Bn14G42-01 (418 bp,AJ415642) being typical of the Ty3 family ofelements, based on those found in Saccharomycescereviseae, and Bn21G50-04 (514 bp, AJ421230),which shows similarity to the Arabidopsis Athila1family (Pelissier et al., 1995). The LINE used inthis study was Bo6L41-06 (416 bp, AJ417795).Further data about diversity and isolation of these,and other elements, in Brassica species are givenby Alix and Heslop-Harrison (2004) and theEMBL database.

BAC library analysis

A BAC library consisting of 26,112 clones with anaverage insert size of 110 kb was constructed fromB. oleracea var. alboglabra A12DHd high molecu-lar weight DNA. The library was made using aHindIII partial digest cloned into the pBeloBAC11vector using TAMU protocols (http://hbz.tamu.edu/bacindex.html, or http://www.brassica.info/ukbrc/kingbrassica/downloadarea/BAC%20libra-ry_1.htm for further details). Colony filters wereprepared using a BioRobotics microgrid TASrobot to array 18,432 colonies (48 microtitre plates)per 22�22 cm membrane. In this study 75% of thefull library was used (19,584 clones).

For BAC library screening, probes werelabelled with [a-32P]dCTP by random priming(Amersham Oligolabelling kit) and purified onSephadex columns. The prehybridization buffercontained 4� SSC, 1% SDS (w/v), 10� Den-hart’s solution (1% polyvinylpyrrolidone (w/v),1% ficol (w/v), plus 1% BSA (w/v)) and 80 lg/ml of sheared salmon sperm DNA. After aprehybridization of 4 h at 55 �C, the labelled

probe was added before hybridization overnightat 55 �C. The post-hybridization washing condi-tions were 5� SSC – 0.1% SDS for 5 min atroom temperature and for 30 min at 55 �C, thentwice for 30 min at 55 �C with 3� SSC – 0.1%SDS. Film exposure times were approximately3 days. When scoring the BAC library hits onautoradiographs, all signals stronger than back-ground were scored.

The coincidence of hits between accumulated‘‘BoB’’ characterisation data from many researchprojects and the retroelement hits obtained wasqueried in search of emergent trends.

Sequence analysis

In order to put our BAC library screen intocontext with other estimates of copy number, wecarried out sequence analysis in a similar mannerto that of Zhang and Wessler (2004). We chose tocarry out BLASTN analysis using WU-BLAST 2.0software (Gish 2005) rather than TBLASTN inorder to maintain comparability with our BAClibrary screening results. A relatively stringent Evalue of 1e-10 and a less stringent value of 1e-2were used in the searches. We analysed each of ourfive retroelement sequences against the B. oleraceagenome survey sequences (0.5� genome coverage)available from The Institute for GenomicResearch (TIGR – preliminary sequence dataobtained from http://www.tigr.org) and againstthe complete Arabidopsis sequence. The formulapresented in Zhang and Wessler (2004) was used togenerate genome copy number estimates from ourBLASTN results using the number of hits andlength of retroelement sequences.

We analysed TE distribution and frequencyusing a combination of GeneMark.hmm version2.4 using the Arabidopsis model (http://opal.biology.gatech.edu/GeneMark/ Lukashin and Borod-ovsky, 1998) and BLASTN on seven B. oleraceaBAC sequences available to us. These BACs aredispersed around the B. oleracea genome;BoB037N21 (AY350710/AY028608 Mayfieldet al., 2001; Fiebig et al., 2004), BoB028L16,BoB048N13 and BoB029L16 (Ryder, Barker,King, unpublished) and three BACs from a differ-ent B. oleracea BAC library B21H13, B21F5 andB19N3 (AC122543 (Gao et al., 2004), AC152123and AC149635).

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Chromosomal in situ hybridization

Chromosome spreads were prepared from root tipsof B. oleracea var. alboglabra A12DHd (genotypeused to construct the BAC library) and B. rapa var.rapifera ‘Chicon’. Two-day old germinating seed-lings were synchronized by overnight refrigerationat 4 �C, followed by a further 24 h growth at 25 �C.Metaphases were accumulated by incubatingexcised roots in 2 mM 8-hydroxyquinoline for 2 hat 25 �C and a further 2 h at 4 �C. Material wasfixed in fresh fixative (3:1 ethanol:acetic acid) for2 days at room temperature, and then stored in70% ethanol at)20 �C.After rinsing, root tips werecut and placed in enzyme solution (3% pectinase (v/v)/2% cellulase, in 6 mM tri-sodium citrate/4 mMcitric acid, pH 4.6) at 37 �C for 30 min. Root tipswere then placed in distilled water for at least30 min and spread on an acid-cleaned slide with adrop of fixative using forceps. Slides were air-driedand stored at )20 �C.

Methods for in situ hybridization essentiallyfollowed Schwarzacher and Heslop-Harrison(2000). The reverse transcriptase probes werelabelled with biotin- and digoxigenin-dUTP(Roche) using the random priming DNA labellingkit (Gibco BRL) and then purified with WizardDNA clean-up system (Promega). The hybridiza-tion mixture consisted of 50% formamide, 2�SSC, 10% dextran sulfate, 0.125% SDS,0.125 mM EDTA, 1 lg of salmon sperm DNA,

and 10–15 ng of each labelled probe, in a finalvolume of 40 ll per slide. This mix was denaturedat 75 �C for 10 min, cooled on ice for 10 min andapplied to the slides; slides and probes weredenatured together at 70 �C for 8 min. Hybridiza-tion was performed overnight at 37 �C beforewashing. The most stringent post-hybridizationwash was at 42 �C in 20% formamide – 0.1� SSC.Hybridization sites were detected using antidigox-igenin conjugated to FITC (Roche) and avidinconjugated to Alexa 594 (Molecular Probes), andchromosomes were counterstained with DAPI.Slides were mounted in AF1 medium (CitifluorLtd) and photographed using an Optronics CCDcamera on a Zeiss Axiophot epifluorescencemicroscope. The images were processed andprinted from Adobe Photoshop 5 using onlycontrast and brightness functions affecting thewhole image equally.

Results

BAC library characterization

An empirical demonstration of predicted genomerepresentation for the ‘‘BoB’’ library is shown inTable 1. The average value of 3.7� genome cover-age is representative of the whole library; for thisstudy we used 75% of the whole library, whichconsequently represents 2.8� genome coverage.

Table 1. Empirical determination of redundancy for the ‘‘BoB’’ BAC library.

Probe/gene Number of ‘‘BoB’’ BAC clones hybridising A12DHd genome copy number Estimated redundancy

BoAp1 11 3a, b, c, d 3.7

BoLfy 6 2b, c 3

BoFul 5 3b, c 1.7

BoRGL-Va 3 1b, d 3

pO123 9 2a 4.5

p0152 11 4b, c 2.75

pR6 21 4a 5.25

pR72 9 2a 4.5

pR94 8 2a 4

pW134 5 1e 5

pW146 6 2a 3

Average estimated redundancy of ‘‘BoB’’ BAC library 3.7

Data for 11 low copy number probes and their deduced representation in the ‘‘BoB’’ library.a Estimated by RFLP analysis. Each distinct band in A12DHd genomic DNA is taken to represent a single genome copy except wheresegregation of bands in an A12DHd mapping population suggests otherwise.b Genomic locations of BACs verified by FISH analysis.c Ascertained by fingerprint analysis.d Ascertained by pcr fragment analysis and sequencing.e Probe used directly as FISH probe without Cot1 and revealed a single signal in the A12DHd genome (Howell et al, 2002).

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Retrotransposon hybridization

Numbers of genomic insertion sites for the fivedifferent retrotransposons in the B. oleracea gen-ome were estimated by examining the presence ofthese elements in a portion of a BAC libraryrepresenting 2.8� genome coverage. As hybridiza-tion scores did not distinguish between BACs withmore than one copy of a probe and those with asingle copy, only estimation of genomic sites andnot of copy numbers could be made. Figure 1shows a section of the BAC filters hybridized withthe copia and gypsy clones. Table 2 shows thenumber of BACs hybridising to each probe. Over-all, 10% of the BACs included retrotransposonshomologous to one or more of the probes, and only0.32% of the clones hybridized to two or moredifferent probes (63 BAC clones from the 19.500BACs analysed – Table 2). As detailed in Table 3,sequencing has been carried out on four ‘‘BoB’’BACs (BoB028L16, BoB029L16, BoB037N21 andBoB048N13). BLASTN analysis of these sequencesverified the results obtained for the retrotransposonlibrary screenings. Assuming the B. oleraceagenome is fully represented in the BAC library, wepredict c. 90 and c. 320 genomic insertion sites forthe two copia-like retrotransposons. With only nineBACs appearing to contain both types of copiaelement (expectation 12), there was no evidence forclustering or repulsion (the difference is not signif-icant with v2=0.7183 whereas the v2 value withthree degrees of freedom and a=0.05 is 7.8147).Weestimate there to be 130 insertion sites in theB. oleracea genome for the Athila-related gypsyelement Bn21G50-04, and 160 insertion sites for thegypsy element Bn14G42-01. Twenty-one BACshybridized to both gypsy probes (expectation 8);this difference is statistically highly significant, with

v2=20.3487whereas the v2 value with three degreesof freedom and a=0.05 is 7.8147, rejecting thehypothesis of no clustering (P<0.00014). Gypsyand copia elements tended to be present on differentBACs, suggesting that each may have a distinctdistribution (Table 2). Only three BACs hybridizedto the LINE probe, which indicates a low copynumber of this specific LINE with a maximum ofthree location sites in the B. oleracea genome.Southern hybridizations were carried out using theLINE RT clone Bo6L41-06 as a probe on EcoRI-restricted DNAs from the three BACs whichhybridized to the LINE probe when screening the‘‘BoB’’ BAC library; one to three major bands wereobtained confirming the presence of the LINE in allthree BACs (Figure 2). In addition, low copynumber of this specific LINE in the B. oleraceagenome was supported by obtaining only very faintSouthern hybridization signals after probing thecorresponding RT clone on EcoRI-restricted geno-mic DNA from B. oleracea var. alboglabraA12DHd (genotype used to construct the BAClibrary) (data not shown).

By querying the nature of coincidental probehits between the retrotransposon BAC scores andavailable hybridization data for over 100 othersequences such as single/low copy functional genesand housekeeping genes we did not observe anyco-localisation or clustering trends. Scoring datafor the hybridization of the retrotransposonprobes to BACs in the ‘‘BoB’’ library is availableon the website http://www.grc.warwick.ac.uk/brassica/bac_libs.html.

Sequence analysis

Analysis of the five retroelement sequences againstgenome survey sequence gave estimates of between

( p y p ); ( ) y ( p y )

Figure 1. The same section of the BAC filters hybridized with the copia and gypsy RT probes showing 17�8 blocks of 16 colonies

(corresponding to a 4�4 square); colonies are duplicated twice within each 4�4 square, so two hybridizing sites are seen for each

positive BAC in a characteristic orientation, so a total of 1088 BACs (5.6% of the data) are shown. (A) Hybridization with Bo10-

COP-16 (18 positive hybridization pairs); (B) hybridization with Bn14G42-01 (25 positive hybridizations).

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60 and 570 copies in the B. oleracea genome, withno evidence of the LINE. Results of BLASTNanalysis and genome copy number estimates aredetailed in Table 3. The estimates of genome copynumber obtained by sequence analysis are of asimilar order and magnitude as those revealed byBAC library screening. Figures obtained whenanalysing the complete Arabidopsis sequencerevealed figures, which follow the trends presented,by Zhang and Wessler (2004) (data not shown).Comparison of results obtained at different Evalue stringencies reveal relatively large differencesfor gypsy Bn14G42-01. This is indicative of ahigher rate of sequence divergence for this elementthan for the three other LTR retrotransposons.

Gene predictions for the seven BAC sequencesanalysed (594 kb in total length) are presented in

Table 4. The TE genes identified were varied intype and minimal evidence for clustering of likeelements was detected, however there weremarked differences in overall TE numbers ineach BAC. On a per BAC basis, estimations ofTE density range from 1 every 82 to 6 kb. Anaverage estimate of 1 TE every 19 kb is obtainedwhen taking this data set as a whole. Theexistence of gene-enriched BACs in oppositionto transposon-rich BACs is also clearly illustratedin the small set of BAC sequences analysed(Table 4). For instance the BAC B21H13 con-tains 23 gene-coding sequences (gene density of 1gene every 4.4 kb) with no TE sequence in itwhile BoB048N13 shows a low gene-density of 1gene every 39.5 kb but a significant enrichment inTEs with the presence of 7 transposon-genes.

Table 3. Summary of results obtained by BLASTN comparison at two different E values of our five B. oleracea retrotransposonRT sequences to the 0.5� coverage set of B. oleracea genome survey sequences from TIGR.

Family RT sequence BLASTN

hit at 1e-10

Genome copy

number estimateaBLASTN hits

at 1e-2

Genome copy

number estimatea

Ty1-copia Bo10COP-16 AJ414054 27 58 31 66

Ty1-copia Bo10COP-18 AJ414055 250 538 265 570

Ty3-gypsy Bn14G42-01 AJ415642 111 205 225 415

Athila-like Bn21G50-04 AJ421230 212 359 246 417

LINE Bo6L41-06 AJ417795 0 0 0 0

a Calculated taking into account length of sequences being compared, probability that 1 hit=1 element and relative genome coverage.

Table 2. Summary of results obtained by screening the ‘‘BoB’’ BAC library with five retrotransposon probes.

Class Family Retrotransposon

probe

Total

number

Expectationa Per genome

equivalentb

LTR Ty1/copia Bo10COP-16 258 92

Ty1/copia Bo10COP-18 894 319

Ty3/gypsy Bn14G42-01 439 157

Athila-like Bn21G50-04 368 131

Non-LTR LINE Bo6L41-06 3 1

Hybridizing to both copia probes 9 12 3

Hybridizing to both gypsy probes 21 8 8

Copia/gypsy cohybridization:

Bo10COP-16 and Bn14G42-01 2 6 1

Bo10COP-16 and Bn21G50-04 1 5 1

Bo10COP-18 and Bn14G42-01 18 20 6

Bo10COP-18 and Bn21G50-04 11 17 4

LINE /gypsy or copia cohybridization: 0 0 0

Total BACs analysed 19500 6964

Total BACs to one probe 1835 655

Total BACs to two or more probes: 63 68 23

a Expectation calculated on the basis of the independent distribution of the five retrotransposons as: (probe 1 hits)�(probe 2hits)�� � ��(probe n hits)/(total clones analysed)n-1; rounded values are shown but exact values were used for statistics.b Per genome equivalent calculated as: probe hits/2.8 (2.8=empirically determined ‘� genome coverage of BACs screened’).

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Only the BAC sequence B19N3 contains a 1:1ratio of gene:transposon sequences. Interestinglythe relative numbers of class I retrotransposonsvs. class II DNA transposons is not entirelyconsistent with current estimates for wholegenome of B. oleracea (Zhang and Wessler,2004). The seven BACs analysed here have beensequenced because they contain coding genes ofinterest and our results are thus in accordancewith the preferential insertion of class II TEs intogenic regions (Zhang et al., 2000; Fu et al., 2001;Walbot and Petrov, 2001).

In situ hybridization

FISH was used to investigate the physical distribu-tion of the two copia retrotransposons (Figure 3)and the Athila-like gypsy retrotransposon(Figure 4) along the chromosomes of B. oleracea.The 410 bp-long RT probe corresponding to theLINEwas not long enough to be efficient in locatingthis low copy non-LTR retroelement and was thusnot cytogenetically analysed. The copia probeBo10COP-16 and the Athila-like gypsy probeBn21G50-04 showed multiple hybridization signals

p p p g p y

Figure 2. Southern hybridization of the LINE RT clone Bo6L41-06 to EcoRI-restricted DNA from the three BACs identified as

homologous by the library screen. (A) Size separated DNA showing BAC fingerprints; (B) hybridization showing one to three ma-

jor bands homologous to the probe. Two BACs (lanes 2 and 3) are similar indicating they are substantially the same in the library,

while the third BAC (lane 1) may or may not overlap. From left to right: ‘‘BoB’’ BACs BoB008A09, BoB015J11 and BoB044F08.

Lane 4: 1 kb ladder.

Table 4. Predicted gene content for seven B. oleracea BAC sequences.

BAC Name Library Length

(bp)

Non-TE

gene

count

Total TE

gene count

Retrotransposon

gene count

Transposon

gene count

Non-transposon

gene density

(1 every nkb)a

Total TE

gene density

(1 every nkb)a

BoB028L01 BoB 109,794 9 5 3 2 12.2 kb 21.9 kb

BoB029L16 BoB 86,024 3 10 4 6 28.6 kb 8.6 kb

BoB048N13 BoB 39,495 1 7 4 3 39.5 kb 5.6 kb

BoB037N21 BoB 78,346 15 1 1 0 5.2 kb 78.3 kb

B19N3 Early Big-10 96,718 8 7 5 2 12.1 kb 13.8 kb

B21F5 Early Big-10 82,329 12 1 0 1 6.9 kb 82.3 kb

B21H13 Early Big-10 101,563 23 0 0 0 4.4 kb –

‘TE’ for transposable element.a Associated estimates of non-transposon gene and TE gene density for each BAC have been calculated.

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on all eighteen chromosomes of B. oleracea, asdouble dots on both chromatids, while the othercopia probe Bo10COP-18 displayed more diffusehybridization signals on sixteen chromosomes withone chromosome pair displaying no hybridizationsignal (Figure 3D and E). Bo10COP-16 showedhybridization to multiple sites dispersed along thefull length of chromosomes. Bo10COP-18 and theAthila-like gypsy element gave hybridization sig-nals that were distributed along chromosomes witha strong tendency to concentrate in broad centro-meric regions; similar hybridization patterns wereobtained for the Ty3-gypsy probe Bn14G42-01 onB. rapa (AA, 2n=2x=20) chromosomes (Fig-ure 5). Analysis of the distribution of theAthila-like gypsy element in interphase nucleidemonstrated that some of the hybridizationsignals co-localize with DAPI chromocentres thatcorrespond to pericentromeric regions (data notshown). The number of chromosome spreadsanalysed was between 3 and 7 for the different RTprobes surveyed.

Discussion

The results from BAC library screening andBLASTN analysis of B. oleracea genome surveysequences with retrotransposon probes from thecopia and gypsy families showed that each probesequence is representative of between 60 and 570

copies which are present at multiple sites over theB. oleracea genome (Tables 2 and 3). All theanalysis methods we have used predict thatthe LINE sequence investigated here is present ata very low copy number in this genome. A resultthat is in accordance with previous Southernhybridization data (Alix and Heslop-Harrison,2004). LINEs constitute the most ancient class ofretrotransposons in plants, and sequence diver-gence and low copy numbers of each LINEsubfamily are typical features of these repetitiveelements (Schmidt, 1999; Lavie et al., 2002; Versh-inin et al., 2002; Zhang and Wessler, 2004), theonly known exception being the abundant del2LINE element of lily (Leeton and Smyth, 1993).Isolation of LINEs using degenerate PCR primersled us to isolate only a small subset of the BrassicaLINE subfamilies (Alix and Heslop-Harrison,2004) and based on homology (hybridizationexperiments and search for sequence identity usingBLASTN) the LINE RT probe used here identifiedonly one specific LINE subfamily, illustrating thatdivergence of LINEs is so pronounced that LINEsequences do not cross hybridize with each other.

The GSS sequences were made from clones,which had been mechanically sheared. The BAClibrary was made by partial restriction enzymedigestion hence may be skewed in its representation– particularly at repetitive regions. Viewed togetherthese approaches give powerful insights into thedistribution of these elements in the Brassica

Figure 3. In situ hybridization of the copias Bo10COP-16 (A, B, C) and Bo10COP-18 (D, E, F, G) to metaphase chromosomes of

B. oleracea. The Bo10COP-16 probe is labelled with biotin and detected by Alexa 564 (red signal) while the Bo10COP-18 probe is

labelled with digoxigenin and detected by FITC fluorescence (green signal); chromosomes are counterstained with DAPI (blue

signal). (A) Complete metaphase plate probed with Bo10COP-16 showing multiple sites of hybridization dispersed all along

the chromosomes with a tendency to hybridize subtelomeric regions. (B, C) illustrate two chromosomes from another metaphase

plate with distinct double dots at the end of sister chromatids. (D, E) Complete prometaphase plate probed with Bo10COP-18

showing preferential centromeric sites of hybridization on sixteen chromosomes, one chromosome pair displaying no hybridization

signal (arrows, E); (F, G) detail of hybridization on two chromosomes.

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genome. The average size of a copia retrotranspo-son is 7 kb (Kumar and Bennetzen, 1999), andgenome size of B. oleracea is 650 Mbp. The copia

retroelements studied here represent thereforebetween 0.44% (=7 kb�(90+320) hybridizedBACs/650,000 kb with the hypothesis of oneBAC containing one single copy of the copiaanalysed) and 0.69% of the haploid B. oleraceagenome (=7 kb�(570+70) hits/650,000 kb),based on BAC library screening and GSS analysis,respectively. For the gypsy group, taking anaverage size of 10 kb (Kumar and Bennetzen,1999), the two elements represent between 0.45%(10�(160+130)/650,000), assuming that eachhybridization of an RT domain probe is represen-tative of a single complete element, and 1.29% ofthe haploid genome (10�(420+420)/650,000). Theretrotransposon families that we have surveyed inour present work represent therefore only a smallpart of the B. oleracea genome, and our currentstudy conducted on specific retrotransposon fam-ilies supports Zhang and Wessler (2004) whorevealed that no massive proliferation of one ormore retrotransposon family has occurred in

Figure 5. In situ hybridization of the Ty3-gypsy-like clone

Bn14G42-01 to metaphase chromosomes of B. rapa. The

probe is labelled with biotin and detected by Alexa 594 fluo-

rescence (red signal); chromosomes are counterstained with

DAPI (blue signal). The probe gave hybridization signals

along chromosomes with a clear tendency to concentrate as

clusters in pericentromeric regions as illustrated by the red

dots. Here are shown three different B. rapa chromosomes

displaying the main patterns of hybridization observed for

Bn14G42-01.

Figure 4. In situ hybridization of the Athila-like clone Bn21G50-04 to metaphase chromosomes of B. oleracea. The probe is

labelled with digoxigenin and detected by FITC fluorescence (green signal); chromosomes are counterstained with DAPI (blue

signal). (A) Complete metaphase plate showing multiple sites of hybridization on all eighteen chromosomes; (B, C, D) chromo-

somes from another metaphase plate illustrating the tendency of Athila elements to concentrate in pericentromeric regions. Red

dots (B, C, D) correspond to sites of hybridization of the copia probe Bo10COP-16 (Figure 3).

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B. oleracea with the most abundant family con-taining around 140 copies. In other words, if Zhangand Wessler (2004) have estimated that total class ITEs comprise up to 14% of the B. oleracea genome,they have demonstrated that this fraction is actu-ally composed of numerous specific class I elementsamplified to relatively low copy number. This is inaccordance with our analysis of prevalence anddistribution of four specific retrotransposon sub-families in the B. oleracea genome presented here.The sequence analysis of seven BACs has beenparticularly informative about the global structureof B. oleracea genome concerning gene and TEdistributions. One major result is the identificationof nearly equilibrated ratios of class I:class IIelements in genic regions, although class II DNAelements account for 6% of the whole B. oleraceagenome or 2.5 times less than class I RNA elements(Zhang and Wessler, 2004), demonstrating thattransposons preferentially target genic regions.

At the chromosomal level, FISH showed indi-vidual elements were dispersed over the chromo-somes with slight clustering trends revealed bydistinct pairs of dots being observed on chromat-ids (eg. Bo10COP-16). Bo10COP-18, Bn21G50-04and Bn14G42-01 had a tendency to locate in broadpericentromeric regions, where we know there aretandemly repeated sequences (Harrison andHeslop-Harrison, 1995). In addition, our in situresults are in accordance with the recently pub-lished work concerning the integration of thecytogenetic and genetic linkage maps of B. olera-cea (Howell et al., 2002): Bo10COP-18 hybridizedto the BAC clone BoB061G14 which has beendemonstrated to hybridise to pericentromeric het-erochromatin of six chromosomes (Figure 2 inHowell et al., 2002). The fact that Bn14G42-01hybridized to BAC clones BoB035N04 andBoB011O13 which have both been located closeto centromeric regions of different B. oleraceachromosomes, (Figure 2 in Howell et al., 2002)suggests that the distribution pattern observed onB. rapa chromosomes for Bn14G42-01 (Figure 5)would also be observed in B. oleracea.

The gypsy retrotransposon data obtained fromthe BAC library screening using the two differentgypsy-type probes supports a tendency towardsclustering and in situ hybridization suggests pref-erential insertion sites in pericentromeric regions.These gypsy retrotransposons may correspond totypical centromericMetaviridae elements that have

been demonstrated to be major components of therepetitive fraction of centromeres of differentspecies (Presting et al., 1998; Hudakova et al.,2001; Kurata et al., 2002; Peterson-Burch et al.,2004). However, it has also been observed thatgypsy retrotransposons can be widely distributedover chromosomes (Vershinin et al., 2002). Athilahas been demonstrated to be concentrated in thepericentromeric heterochromatin of Arabidopsis,where it is associated with the major 180-bpcentromeric satellite DNA (Pelissier et al., 1996;Peterson-Burch et al., 2004). The present workdemonstrates similar distribution of the Athila-likegypsy element in B. oleracea with a tendency to bemore widely distributed in the genome; this limitedbut clear dispersion of Athila retroelements fromthe centromere observed in the B. oleracea genomemay reflect the amplification of Athila-likeelements that has occurred since divergence fromthe common ancestor of the Brassicaceae.

Bo10COP-16 displays a typical dispersionpattern of copia retrotransposons, they have oftenbeen shown to be dispersed along the chromosomeswith trends of clustering in some interstitial chro-mosomal regions (Brandes et al., 1997; Kuiperset al., 1998; Benabdelmouna and Darmency,2003). However, Bo10COP-18 displays a contrast-ing distribution pattern with preferential insertionsin centromeric regions that have been rarelyobserved for Pseudoviridae (Brandes et al., 1997;Francki, 2001). In addition to high sequenceheterogeneity that we have previously demon-strated (Alix and Heslop-Harrison, 2004), the twonon-homologous copia retrotransposons used inthis study show dissimilarities in genomic distribu-tion and organization. Flavell et al. (1992) demon-strated the high heterogeneity in the copia group ofretrotransposons for eight different plant genomes,by comparing the various nucleotide sequences ofcopia RT domains amplified by PCR; similarresults were obtained from the analysis of copiaretrotransposons in Arabidopsis (Terol et al., 2001)or cereal species (Benabdelmouna and Darmency,2003). We conclude that the different copia ele-ments either have insertion site preferences, or areremoved from genomic regions at differing rates.Whichever model is followed, it is interesting thatthere is such a difference in elements, which sharethe same structure and mode of amplification.

The evidence from B. oleracea BAC libraryscreening suggests no strong clustering of retroe-

848

lements from different families, with copia andgypsy clones tending not to occur on the sameBAC. In addition, our in situ hybridization resultsreveal minimal evidence of clustering betweenretrotransposon types. Assuming that the BAClibrary and genome survey sequence we analysed isrepresentative of the whole B. oleracea genome,our data contrast at some points with previousresults in species with larger genomes such asmaize (SanMiguel et al., 1996, genome size1000 Mbp) and Hordeum (Vershinin et al., 2002,genome size 5500 Mbp). These demonstrated thatmany retrotransposons from different families areinserted within each other to form a nestedstructure. In A. thaliana, a species with only 20%of the DNA of B. oleracea, retrotransposons arelargely clustered in the genome, with a particularconcentration around the centromeres (Peterson-Burch et al., 2004). However, detailed analysis ofseven B. oleracea BAC sequences has supportedthe occurrence of transposon-enriched genomicregions, in contrast to gene-rich regions, with classI and class II TEs being inserted in the samegenomic region. This seems to be a commonfeature of plant genomes as this phenomenon hasbeen observed in different plant species includingArabidopsis (Miura et al., 2004), maize (SanMig-uel et al., 1996, 1998; for review Walbot andPetrov, 2001) and wheat (Anderson et al., 2003).

In conclusion, the retrotransposons represent asignificant component of the Brassica oleraceagenome and their distribution gives an insight intothe evolution and variation present within boththese sequences and the genome as a whole.Within the Brassicas, these results provide areference to look at many aspects of the large-scale genomic rearrangements and evolution of thegenus under selection pressure. For example,comparisons between species or subspecies anddetermination of ancestral taxa of the tetraploids(e.g. Warwick et al., 2003) give insights into thenature of the diploid species (which seem to have apalaeohexaploid origin). Evidence suggests thatretrotransposons are related to gene transfer andactivation/suppression, particularly followingstress from tissue culture and generation of trans-genic plants. Consequently identification of activeand potentially active elements, sometimes associ-ated with genes, may be valuable: in Brassica,retrotransposons provide useful markers by theirinsertional polymorphisms (Flavell et al., 1998).

Knowledge of the nature of the repetitive DNAfamilies in the genus will not only be helpful inunderstanding the continuity of the A, B and Cgenome gene-pools (Chevre et al., 1997) but alsofurthers our understanding of the relationship ofthese genomes to that of the fully sequencedArabidopsis (Ryder et al., 2001).

Acknowledgements

We are grateful to Dr. S.E. Kubis and Dr. E. Jen-czewski for valuable discussions andDr. T. Schwarzacher for helpful advice. Dr. K.Alix was supported by a fellowship under theOECD Co-operative Research Programme: Bio-logical Resource Management for SustainableAgriculture Systems. We are grateful to BBSRCfor funding GJK, CDR, JM and Guy Barker andthe BBSRC-IGF project for access to hybridiza-tion data. Preliminary sequence data fromB. oleracea was obtained from The Institute forGenomic Research website at http://www.tigr.org. Sequencing of B. oleracea was funded bythe ‘‘National Science Foundation’’.

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