Genetic distribution of Bare–1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP

Genetic distribution of Bare–1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP

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  ORIGINAL PAPER R. Waugh  ·  K. McLean  ·  A. J. FlavellS. R. Pearce  ·  A. Kumar  ·  B. B. T. ThomasW. Powell Genetic distribution of Bare–1-like retrotransposable elementsin the barley genome revealed by sequence-specific amplificationpolymorphisms (S-SAP) Received: 17 July 1996 /Accepted: 20 September 1996 Abstract  Retrotransposons are present in high copynumber in many plant genomes. They show a con-siderable degree of sequence heterogeneity and inser-tional polymorphism, both within and between species.We describe here a polymerase chain reaction (PCR)-based method which exploits this polymorphism for thegeneration of molecular markers in barley. The methodproduces amplified fragments containing a Bare–1-likeretrotransposon long terminal repeat (LTR) sequence atone end and a flanking host restriction site at the other.The level of polymorphism is higher than that revealedby amplified fragment length polymorphism (AFLP) inbarley. Segregation data for 55 fragments, which werepolymorphic in a doubled haploid barley population,were analysed alongside an existing framework of some400 other markers. The markers showed a widespreaddistribution over the seven linkage groups, which isconsistent with the distribution of the Bare–1 class of retrotransposons in the barley genome based on in situhybridisation data. The potential applicability of thismethod to the mapping of other multicopy sequences inplants is discussed. Key words  Bare-1  ·  Retrotransposons ·  Barely  · Linkage analysis  ·  Sequence-Specific AmplificationPolymorphisms (S-SAPs) Introduction Retrotransposons are the commonest class of eukaryotictransposable element. They are separated from othertransposons by their ability to transpose via an RNAintermediate, which they convert to DNA by reversetranscription prior to reinsertion. The retrotransposonsinclude long terminal repeat (LTR) and non-LTR clas-ses (Boeke and Corces 1989; Grandbastien 1992; Smyth1993; Kumar 1996). The most studied group of LTRretrotransposons is the  Ty1 - copia  group, named afterthe best studied elements in  Saccharomyces cerevisiae and  Drosophila melanogaster. Ty1-copia  retrotranspo-sons are also present throughout the plant kingdom(Flavell et al. 1992a, b; Voytas et al. 1992; Hirochika andHirochika 1993; Lindauer et al. 1993). Several of theplant elements have been fully sequenced including Ta1of   Arabidopsis thaliana  (Voytas and Ausubel 1988), Tnt1of tobacco (Grandbastien et al. 1989), Tst1 of potato(Camirand et al. 1990) and Bare–1 of barley (Manninenand Schulman 1993). They are generally present in highcopy number and occupy a considerable proportion of some plant genomes Flavell et al. 1992b; Hirochika et al.1992; Leeton et al. 1993). In addition, retrotransposonsare frequently found in the regions flanking known plantgenes (White et al. 1994).Although a growing body of knowledge is availableon the phylogenetic relationships among the populationsof plant  Ty1-copia  retrotransposons, less is known of thedistribution of these elements within plant genomes.In situ hybridisation of   Ty1-copia  retrotransposonsequences to metaphase chromosomes in sugarbeet(Schmidt et al. 1996) and faba bean (Pearce et al. 1996)has revealed that the  Ty1-copia  elements of these speciesare located throughout the euchromatin and appearedto be mostly absent from heterochromatic regions.Since the development of polymerase chain reaction(PCR) technology (Saiki et al. 1988), a large number of approaches have been developed which allow the de-tection of polymorphism at the DNA level. These in- Mol Gen Genet (1997) 253:687–694  ©  Springer-Verlag 1997Communicated by H. SaedlerR. Waugh ( & )  ·  K. Mclean  ·  A. Kumar  ·  B. B. T. ThomasW. PowellDepartment of Cell and Molecular Genetics,Scottish Crop Research Institute, Invergowrie,Dundee DD2 5DA, UKA. J. Flavell  ·  S. R. PearceDepartment of Biochemistry, University of Dundee,Dundee DD1 4HN, UK  clude sequence-specific methods such as cleaved ampli-fied polymorphisms (CAPs; Konieczny and Ausubel1993) and simple sequence repeats (SSRs; Tautz 1988)and a range of generic approaches such as randomlyamplified polymorphic DNA (RAPDs; Williams et al.1990), arbitrary primed-polymerase chain reaction (AP-PCR; Welsh et al. 1991), DNA amplification finger-printing (DAF; Caetano-Annoles 1993) and randomamplified microsatellite polymorphism (RAMPs; Ziet-kiewicz et al. 1994). The most recent, and arguably oneof the most powerful techniques, is amplified fragmentlength polymorphism (AFLP; Zabeau and Vos 1993).AFLP relies on PCR amplification of a subset of smallrestriction fragments generated by digesting genomicDNA with a frequent and a rare cutting restriction en-donuclease and ligation of known sequence adaptors tothe fragment ends (Vos et al. 1995). The main attractionof AFLP is its high multiplex ratio (Powell et al. 1996)which allows the generation of a large body of in-formation from a relatively small number of individualassays. A few recent publications demonstrate the utilityof AFLP for both saturation mapping (Ballvora et al.1995; Meksem et al. 1995; C.M. Thomas et al. 1995) andgenome-wide linkage mapping (van Eck et al. 1995). Inbarley, studies by ourselves (Powell et al. 1996; Waugh etal. 1996) and others (Becker et al. 1995) have shownthat, while AFLP will be a very powerful approach,scope does exist for improvement on a number of fronts.For example, the level of detectable polymorphism isrelatively low and few markers are codominant. In ad-dition, preliminary observations suggest that AFLPsgenerated after digestion of the DNA with the re-commended enzyme pair of   Eco RI and  Mse I are fre-quently clustered in centromeric regions and at the edgesof genomic segments which are heavily mapped withRFLPs (Becker et al. 1995; Waugh et al. 1996).The ubiquitous distribution, high copy number andwidespread chromosomal dispersion of the  Ty1-copia group of retrotransposons in plants provides excellentpotential for developing a multiplex DNA-based markersystem (Kumar 1996). Here we describe the use of Bare– 1-likeretroelementLTRsequencesinbarleyfordetectingDNA polymorphisms based on the location of the ret-roelements relative to adjacent restriction endonucleasesites. The usefulness of this system has been demon-strated by following the inheritance of a number of theseDNA polymorphisms relative to a range of other mar-kers in a doubled haploid (DH) barley population. Thishas allowed us to show that the genetical distribution of Bare–1 elements in barley correlates well with the phy-sical distribution, as revealed by in situ hybridisation. Materials and methods MaterialsA DH barley population (B  ×  E) derived from the F 1  of a crossbetween the cv Blenheim and breeding line E224/3, which haspreviously been extensively mapped with RFLPs, RAPDs andAFLPs (W.T.B. Thomas et al. 1995), was used for all studies. DNAwas extracted from 3-week-old plants according to Saghai-Maroof et al. (1984).In situ hybridisationSeeds of   Hordeum vulgare  cv Blenheim were germinated by placingon moist filter paper. Root tips 1 cm in length were cut off andplaced on 0.5% colchicine for 4 h, then fixed in 3:1 methanol:aceticacid. Fixed root tips were washed in enzyme buffer (6 mM sodiumcitrate, 4 mM citric acid, pH 4.6) followed by digestion with cel-lulase and pectinase (1.6% cellulase, Calbiochem; 0.4% cellulase,Onasaka; 20% pectinase, Sigma) at 37 °  C for 80 min. Squasheswere made in 45% acetic acid and coverslips removed after freezingon dry ice. In situ hybridisation followed the technique of Leitch etal. (1994). Briefly, 1   g Bare–1 DNA was labelled with biotinylateddUTP (Boehringer Mannheim) by the random primed method.Chromosome preparations were pretreated with RNase (100 mg/mlfor 1 h at 37 °  C). The hybridisation mixture consisted of 50%formamide, 10% dextran sulphate, 0.1–0.5% SDS, autoclaved sal-mon sperm DNA (25–100 ng/l), 2  ×  SSC with 50 ng/slide of labelledprobe. The probe mix was denatured at 70 °  C for 10 min and heldon ice for 5 min before being added to the slides. Probe and root tippreparations were then denatured at 80 °  C for 5 min, before hy-bridisation overnight at 37 °  C. Post-hybridisation washes werecarried out, the most stringent being 20% formamide in 0.1  ×  SSCat 37 °  C. Sites of hybridisation were detected using fluoresceinisothiocyanate extravidin-(FITC) conjugate (Sigma). The chromo-somes were counterstained with 2 g/ml 4 ′ , 6-diamidino-2-pheny-lindole (DAPI) in McIlvaines citric buffer pH 7, before examinationby epifluorescent light microscopy.Sequence-specific amplification polymorphism (S-SAP)marker analysisTotal DNA (1.0   g) was restricted with  Pst I and  Mse I (5 U of each)for 1 h in 1  ×  RL buffer (10 mM TRIS-acetate pH 7.5, 10 mMmagnesium acetate, 50 mM potassium acetate, 5 mM DTT, 5 ng/  lBSA) (Vos et al. 1995) in a total volume of 20   l. Template DNAwas prepared by adding 4   l of a ligation mix [4 pmol  Mse I (5 ′ -GACGATGAGTCCTGAG-3 ′  plus 5 ′ -TACTCAGGACTCAT-3 ′ ),2 pmol  Pst I adaptor (5 ′ -CTCGTAGACTGCGTACATGCA-3 ′ plus 5 ′ -TGTACGCAGTCTAC-3 ′ ), 1.2 mM ATP, 1  ×  RL bufferand 0.5 U T4 DNA ligase] and the samples incubated for 3 h at37 °  C, then stored at 4 °  C. The template DNA was then pre-amplified to select and bulk restriction fragments of the correct sizeand configuration using primers homologous to the adaptor se-quences (P: 5 ′ -GACTGCGTACATGCAG-3 ′ ; M: 5 ′ -GATGAGT-CCTGAGTA-3 ′ ) in 25   l reactions containing 0.75   l templateDNA, 75 ng P and 75 ng M oligonucleotides, 0.2 mM dNTPs,1  ×  PCR buffer (Perkin Elmer) and 1 U  Taq  DNA polymerase(Perkin Elmer). PCR on a Perkin Elmer 9600 instrument comprised30 cycles of 94  ° C (30 s), 60  ° C (30 s), 70  ° C (1 min). Then, 55   lTO.1E (10 mM TRIS-HCl, pH 8.0, 0.1 mM EDTA) was thenadded and the preamplified DNA stored at 4 °  C. The Bare–1-likeLTR oligonucleotide (Bare-1-LTR: 5 ′ -CTAGGGCATAATTCC-AACA-3 ′ ) was end-labelled by combining 0.1   l   -[ 33 P]ATP(3000 Ci/mmol), 0.1   l 10  ×  T4 buffer (0.25 M TRIS-HCl pH 7.5,0.1 M MgCl 2 , 50 mM DTT, 5 mM spermidine), 0.134   l Bare–1-LTR (50 ng/  l stock), 0.25 U T4 kinase (0.025   l) and 0.641   lsterile distilled H 2 O per subsequent reaction. Each selective am-plification reaction contained 1   l  33 P-labelled Bare–1-LTR, 0.5   lunlabelled Bare–1-LTR (50 ng/  l), 0.6   l P ( )  or M ( )  selectiveamplification primer (50 ng/  l stocks), 2   l dNTP mix (2 mMstocks), 2   l dNTP mix (2 mM stocks), 2   l 10  ×  PCR buffer, 0.5 U Taq  DNA polymerase (Perkin Elmer), 11.8   l distilled H 2 O and 2   lpreamplified DNA. M ( )  and P ( )  primers had the same sequence asthe P and M primers given above but included one to three addi-tional selective nucleotides at the 3 ′  end. The primers used were M,M (C) , M (AC) , M (ACA)  P, P (C) , P (CC) , P (CG) , P (GT) , P (TC) , P (AAT) ,688  P (AAA) , P (ACG) , P (AGC) , P (ATT) , P (CGA) , P (GGA) , P (GTA) , P (TAC)  andP (TCG) . The touchdown PCR protocol of Vos et al. (1995) wasfollowed exactly.Gel electrophoresis/autoradiographyAfter PCR, 20   l gel loading buffer (94% formamide, 10 mMEDTA, 0.5 mg/ml xylene cyanol FF, 0.5 mg/ml bromophenol blue)was added to each tube and the samples denatured by incubation at90  ° C for 5 min and then placed directly on ice. A 3.8-  l aliquot of each sample was loaded onto 6% denaturing polyacrylamide gels(40  ×  30  ×  0.04 cm) (Sambrook et al. 1989) which had been prerunat 80 W for 30 min. Samples were electrophoresed for 1 h 45 min at80 W constant power alongside an M13 sequencing marker. Gelswere dried directly onto the glass plates and exposed to KodakX-Omat film for 1–5 days at room temperature.Linkage analysisSegregating Bare–1-LTR-derived marker data were entered along-side existing marker data for the B  ×  E population and analysedusing Joinmap V 2.0 (Stam and van Ooijen 1995). Parameters wereset for F 1  DH-derived progeny using  a  for markers derived fromBlenheim and  b  for markers from E224/3. Mapping was carried outas described by Powell et al. (1996) except that the results from thethird cycle of JOINMAP were used to examine the distribution of the S-SAPs. Results Physical distribution of Bare–1-like retrotransposableelements in the barley genomeAn important feature of a molecular assay system whichwill affect its general application is the overall distribu-tion of markers in the genome of the species understudy. To determine the distribution of Bare–1 elementsin the barley genome, a Bare–1 probe was hybridised tometaphase chromosome spreads from barley root tippreparations. Figure 1 shows the sites of Bare–1 hy-bridisation. Bare–1 elements are present at high copynumber and are distributed throughout the barley gen-ome. A less intense staining is apparent at both cen-tromeric and nucleolar organiser regions. A controlexperiment using a portion of the reverse transcriptasedomain of a different barley retrotransposon gave muchreduced staining throughout the genome (not shown).These results are consistent with those recently reportedby Suoniemi et al. (1996).Bare–1-LTR-derived sequence-specific amplificationpolymorphisms (S-SAPs)The observed physical distribution of Bare–1 elementssuggested that the relatively highly conserved sequencesof these elements could be exploited to develop a gen-ome-wide multiplex assay for revealing molecular poly-morphisms between related individuals. There are anumber of possible approaches to developing a PCR-based marker from retrotransposable elements, exploit-ing the inherent variation either within the element itself or the variation in the DNA flanking its site of insertion.We chose the latter approach by modifying the recentlydescribed AFLP technique (Vos et al. 1995) to detectBare–1-like elements. The procedure is outlined inTable 1. The initial steps were identical to the AFLPprotocol. However, the selective amplification employeda single adaptor-homologous primer, along with a Bare– 1-derived radiolabelled primer originating from thehighly conserved terminus of the LTR (Manninen andSchulman 1993). The P ( )  or M ( )  adaptor-homologousprimers carried 0, 1, 2 or 3 selective nucleotides at their3 ′  ends. Figure 2a shows the effect of adding selectivenucleotides to an M ( )  primer when used in combinationwith labelled Bare–1-LTR on two barley lines, Blenheim Fig. 1 a  Metaphase spread of barley chromosomes counterstainedwith 4 ′ ,6-diamidino-2-phenylindole (DAP).  b  Metaphase chromo-somes showing distributions of hybridised biotinylated Bare–1elements visualised by binding to streptavidin fluorescein isothiocya-nate-(FITC) conjugates. The Bare–1 elements appear to be present onall chromosomes with the exception of the centromeric and nucleolarorganiser regions ( arrow ) Table 1  Steps involved in Bare–1-LTR-driven sequence-specificamplification polymorphisms (S-SAP)1. Digest genomic DNA with rare and frequentcutting restriction enzyme2. Ligate on compatible adaptors3. Preamplify prepared template DNA withadaptor-homologous primers4. Selectively amplify with [ 33 P]-labelled sequence-specificoligonucleotide (Bare–1-LTR) and eitherrare or frequent site adaptor homologous oligonucleotide5. Denature and separate in high solution gel689  and E224/3. With no selective nucleotides, a majorproduct is amplified on a less intense background of other fragments. This could be expected because theBare–1-LTR primer can prime amplification both intothe flanking DNA sequence and into the core of theelement itself, as Bare–1 LTRs are direct repeats. Thelargest subclass of Bare–1 elements with a conserved Mse I site within an easily amplifiable distance from theLTR would therefore be expected to generate a majorproduct. The sequenced Bare–1 has an  Mse I site 843 bpinside the 3 ′  LTR. In our case, the major product is ca450 bp, suggesting that it originates from a class of Bare–1 elements which is different from that char-acterised previously. A survey of the published Bare–1sequence revealed 36 sequence motifs within 500 bp of the internal Bare–1 LTR sequence which could poten-tially form an  Mse I site by mutation at a single position.At approximately 450 bp 5 ′  of the internal LTR se-quence, there is a cluster of three potential  Mse I siteswithin a 10-bp window.The addition of up to three selective nucleotides ef-fectively reduces the contribution of this individual classto the overall profile obtained and reduces the number of products to a level which is easily interpreted on a bandpresence/absence basis. Using the P ( )  series of primersproduced broadly similar results, with resolution of in-dividual fragments requiring fewer selective nucleotidesdue to the lower relative frequency of   Pst I restrictionendonuclease sites in the barley genome. Only two po-tential (1 bp mismatch)  Pst I sites were found within the500 bp immediately 5 ′  to the internal LTR sequence,suggesting that the contribution of the Bare–1 elementitself to the overall profile obtained with the P ( ) homologous primers in Bare–1-LTR-driven S-SAP isprobably minor (Fig. 2b). No products were generatedwhen Bare–1-LTR was used on its own. This suggeststhat no (intact) Bare–1 elements exist in the barleygenome in a head-to-head orientation within a distancewhich is readily amplifiable by standard PCR (Fig. 2b).The level of polymorphism detected by Bare–1-LTR-driven S-SAPs is compared to that derived from AFLPusing either  Eco RI (+3)/ Mse I or  Pst I (+3)/ Mse I on thesame parental lines (Table 2). With the primers tested,Bare–1-LTR-driven S-SAP produces fewer products.This is probably a reflection of the selective nucleotidesadded. However, a higher proportion of the S-SAPs arepolymorphic. Fig. 2 a  Effect of one to three additional selective nucleotides on anM-homologous adaptor oligonucleotide used in polymerase chainreaction (PCR) with  33 P-labelled Bare–1-LTR. Addition of selectivenucleotideseffectivelyreducesthenumberofamplificationproductstoa useful number at +3.  Lanes1  Bare–1-LTR + M-homologousoligonucleotide,  2  Bare–1-LTR + M (C) ,  3  Bare–1-LTR + M (AC) ,  4 Bare–1-LTR + M (ACA) .  b  Combination of P (+ 3)  primers with labelledBare–1-LTR oligocucleotide.  Lanes 5  Bare–1-LTR,  6   Bare–1-LTR + P (AAA) ,  7   Bare–1-LTR + P (AAT) ,  8  Bare–1-LTR + P (ACG) , 9  Bare–1-LTR + P (AGC) ,  10  Bare–1-LTR + P (ATT) ,  11  Bare–1-LTR + P (CGA) ,  12  Bare–1-LTR + P (GGA) . ( B   Blenheim,  E   E224/3) Table 2  Comparison of Bare–1-LTR sequence-specific amplification polymorphisms ( S-SAPs ) and amplified fragment length poly-morphisms ( AFLPs ) between Blenheim and E224/3Number of primercombinationsTotal numberof amplifiedproductsNumber of polymorphicproductsProportion of polymorphicproductsAFLP  Eco RI (+3)/ Mse I (+ 3) a 39 92.3 ± 3.9 8.5 ± 1.03 0.092 ± 0.0049 Pst I (+3)/ Mse I (+3) 18 42.67 ± 4.23 8.0 ± 0.68 0.193 ± 0.015S-SAP  Pst I (+3)/Bare–1-LTR 10 36.27 ± 2.71 10.62 ± 1.79 0.26 ± 0.039 a From Powell et al. (1996)690  Genetic distribution of Bare–1-like elements in barleyThe two barley genotypes used to test the S-SAP ap-proach (Blenheim and E224/3) have been used pre-viously to generate a DH population (B  ×  E) which hasin turn been used to construct a genetic linkage map(W. T. B. Thomas et al. 1995; Powell et al. 1996). Toestablish the utility of S-SAPs in genetic linkage studies,we examined the segregation of Bare–1-LTR-derivedamplicons in this population. Six different P ( )  and M ( ) primers used in combination with Bare–1-LTR gener-ated 54 segregating markers (an average of 10.62 perprimer pair). An example of the segregation patternsobserved is given in Fig. 3. At low frequency, productswere observed which segregated in the progeny withinexpected ratios which were not present in either of theparental lines (highlighted in Fig. 3.) This has been ob-served previously in this population with other mole-cular assays (RFLP, RAPD, AFLP) and has beenattributed to residual heterozygosity in one of the par-ental lines used to construct the DH population. Thesewere excluded from the linkage analysis. At very lowfrequency, amplicons which were monomorphic in bothof the parental lines and the progeny were missing froma single individual progeny line (the highlighted case wasthe only clear cut observation). There are several pos-sible explanations for this. Either a genomic rearrange-ment (insertion, deletion or inversion) has occurredwhich has removed the Bare–1-LTR sequence, a pointmutation has occurred in the adjacent  Pst I site or in the5 ′  end of the LTR which abolishes effective primerbinding or the  Pst I site has become methylated in thisline. The exact cause was not determined.Of the 54 S-SAPs segregating in the B  ×  E popula-tion, 48 were mapped to individual linkage groups usingthe existing marker information (Fig. 4). All behaved asdominant markers. The Bare–1-LTR-derived S-SAPsare located on all seven barley linkage groups. Theirdistribution largely reflects that observed for other typesof markers in this population (Powell et al. 1996) andcorrelates well with expectations based on the physicaldistribution of Bare–1 elements revealed by in situ hy-bridisation (Fig. 1). Their inclusion did not result in themerging of any segments from the same group and, withone exception, did not extend beyond the preexistingterminal markers of groups. Discussion The barley genome is estimated to contain upwards of 70000 Bare–1 elements (A. Schulman, personal com-munication), giving enormous scope for the develop-ment of new classes of markers based on Bare–1sequences. Here, using the sequence of Bare–1, we havedeveloped an assay for revealing polymorphism (S-SAP)which uses a combination of AFLP and sequence-spe-cific PCR. The Bare–1 sequence was from the terminal 5 ′ end of the LTR. By using a transposable element se-quence, we hoped to increase the level of polymorphismdetectable in barley to above that revealed by AFLP.While the number of polymorphisms detected per Bare– 1-LTR-driven S-SAP assay was not significantly differ-ent from AFLP in the same population (Powell et al.1996), the proportion of polymorphic products appar-ently was. This will, however, need testing across a widerrange of genotypes before we can confirm this observa-tion.Assuming that all products are derived from Bare–1-like LTR sequences (which was not definitively de-termined), a fraction of the polymorphism revealed will Fig. 3  Segregation of Bare–1-LTR + P (GT) -derived sequence-specificamplification polymorphisms (S-SAPs) in the first 12 progeny of adoubled haploid barley population. A number of bands which arepolymorphic between the parental lines can be seen to segregate in theprogeny. At position  a , a band segregates in the progeny which is notpresent in either of the parental lines. At position b  in the ninth trackfrom the left, a product is absent which is present in both parents andall other progenies691
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