Rapid development of PCR-based genome-specific repetitive DNA junction markers in wheat

TECHNIQUES / TECHNIQUES

Rapid development of PCR-based genome-specific repetitive DNA junction markers in wheat

Humphrey Wanjugi, Devin Coleman-Derr, Naxin Huo, Shahryar F. Kianian,Ming-Cheng Luo, Jiajie Wu, Olin Anderson, and Yong Qiang Gu

Abstract: In hexaploid wheat (Triticum aestivum L.) (AABBDD, C = 17 000 Mb), repeat DNA accounts for *90% of thegenome, of which transposable elements (TEs) constitute 60%–80%. Despite the dynamic evolution of TEs, our previousstudy indicated that the majority of TEs are conserved and collinear between the homologous wheat genomes, based onidentical insertion patterns. In this study, we exploited the unique and abundant TE insertion junction regions identifiedfrom diploid Aegilops tauschii to develop genome-specific repeat DNA junction markers (RJM) for use in hexaploidwheat. In this study, both BAC end and random shotgun sequences were used to search for RJM. Of the 300 RJM primerpairs tested, 269 (90%) amplified single bands from diploid Ae. tauschii. Of these 269 primer pairs, 260 (97%) amplifiedhexaploid wheat and 9 (3%) amplified Ae. tauschii only. Among the RJM primers that amplified hexaploid wheat, 88%were successfully assigned to individual chromosomes of the hexaploid D genome. Among the 38 RJM primers mappedon chromosome 6D, 31 (82%) were unambiguously mapped to delineated bins of the chromosome using various wheat de-letion lines. Our results suggest that the unique RJM derived from the diploid D genome could facilitate genetic, physical,and radiation mapping of the hexaploid wheat D genome.

Key words: wheat genome, genome-specific marker, repeat DNA junction, retrotransposon, genetic and physical mapping.

Resume : Chez le ble hexaploıde (Triticum aestivum L.) (AABBDD, C = 17 000 Mb), l’ADN repete compte pour *90 %du genome et les elements transposables (TE) en constituent 60–80 %. En depit de l’evolution dynamique des TE, les tra-vaux menes anterieurement par ces auteurs indiquent que la majorite des TE sont conserves et co-lineaires entre les chro-mosomes homeologues du ble sur la base de l’observation d’insertions identiques. Dans ce travail, les auteurs ont exploitel’abondance et le caractere unique des insertions chez l’Aegilops tauschii diploıde pour developper chez le ble hexaploıdedes marqueurs specifiques des genomes sur la base des jonctions avec ces ADN repetes (marqueurs RJM pour « repeatDNA junction markers »). Dans ce travail, tant des sequences terminales de clones BAC que des sequences aleatoires ontete utilisees pour rechercher des RJM. Des 300 paires d’amorces RJM testees, 269 (90 %) ont amplifie des bandes uniqueschez l’Ae. tauschii. De ces 269 paires d’amorces, 260 (97 %) ont amplifie chez le ble hexaploıde alors que 3 % n’ont am-plifie que chez l’Ae. tauschii. Parmi les amorces RJM qui ont amplifie chez le ble hexaploıde, 88 % ont ete assignes avecsucces a des chromosomes individuels du genome D. Parmi les 38 amorces RJM situees sur le chromosome 6D, 31(82 %) ont ete assignees clairement a un segment du chromosome 6D a l’aide de diverses lignees portant des deletions.Ces resultats suggerent que les RJM uniques derives du genome D diploıde permettront de faciliter la cartographie gene-tique, physique et de radiation du genome D chez le ble hexaploıde.

Mots-cles : genome du ble, marqueur specifique du genome, jonction avec un ADN repete, retrotransposon, cartographiegenetique et physique.

[Traduit par la Redaction]

Received 30 September 2008. Accepted 6 March 2009. Published on the NRC Research Press Web site at genome.nrc.ca on 28 May2009.

Corresponding Editor: P. Gulick.

H. Wanjugi, D. Coleman-Derr, N. Huo, O. Anderson, and Y.Q. Gu.1 Genomics and Gene Discovery Unit, USDA/ARS WesternRegional Research Center, Albany, CA 94710, USA.S.F. Kianian. Department of Plant Sciences, North Dakota State University, Fargo, ND 58105, USA.M.-C. Luo and J. Wu. Department of Plant Sciences, University of California, Davis, CA 95616, USA.

1Corresponding author (e-mail: [email protected]).

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Genome 52: 576–587 (2009) doi:10.1139/G09-033 Published by NRC Research Press

Introduction

In most plants with complex genomes, transposable ele-ments (TEs) and other repetitive sequences are the majoridentifiable non-genic DNA. TEs have been associated withevolution, specifically the structural and functional alterationof genes and genomes (Kumar and Bennetzen 1999; Devos etal. 2002; Li et al. 2004; Chantret et al. 2005), and are catego-rized into two classes depending on their mode of replicationand transposition. Class I includes RNA transposable ele-ments, or retrotransposons, which replicate via a ‘‘copy-and-paste’’ transposition mechanism such that a single genomiccopy can be the source of numerous new insertions (Kumarand Bennetzen 1999). Class II includes DNA transposons,which replicate via a ‘‘cut-and-paste’’ mechanism (Kumarand Bennetzen 1999). Although both classes of TEs cancause mutations by inserting within or near genes, many in-sertion events are generally neutral to selection and the inser-tion site can be stably retained (Kumar and Bennetzen 1999).

Retrotransposons can be further divided into long terminalrepeat (LTR) and non-LTR retrotransposons. Retrotranspo-sons (especially LTR retrotransposons that terminate in per-fect inverted dinucleotide repeats, usually 5’-TG-3’ and 5’-CA-3’) comprise the bulk of total genomic DNA in plantswith large genomes, such as wheat (Triticum aestivum), bar-ley (Hordeum vulgare), and maize (Zea mays) (SanMigueland Bennetzen 1998; Shirasu et al. 2000; Wicker et al.2001). For example, TEs make up about 80% of wheat nu-clear DNA and consist almost exclusively of LTR retroele-ments (SanMiguel et al. 2002; Wicker et al. 2003a; Gu etal. 2004; Li et al. 2004). DNA transposons can be dividedinto several families. Among them are the CACTA family,which terminate in a conserved inverted 5’-CACTA-3’ and5’-TAGTG-3’ motif (Pereira et al. 1986; Wicker et al.2003a), and the MITEs (miniature inverted terminal re-peats), which have short conserved 5’-TACT-3’ and 5’-AGTA-3’ inverted terminal repeats (Bureau and Wessler1994; Wessler et al. 1995). In general, the dispersion of re-petitive DNA in the genome is random, but insertion hot-spots for TEs in chromosomes of particular plant specieshave been reported (Kumar and Bennetzen 1999). In thewheat genome, LTR retrotransposons are interspersed withinthe intergenic regions, where they form large nested inser-tion structures and cause massive rearrangements, mostlydue to illegitimate or unequal homologous recombination(Devos et al. 2002; Wicker et al. 2003b; Chantret et al.2005). These recombination events often result in truncatedversions of full-length LTR retrotransposons.

The evolution of hexaploid wheat involved the hybridiza-tion of 3 ancestral genomes (A, B, and D) (Kimber andFeldman 1987). The first event was the hybridization of Tri-ticum urartu (AA genome donor) and an unknown BB ge-nome donor of the section Sitopsis of Triticum, resulting ina tetraploid (AABB) (Feldman et al. 1995; Blake et al.1999). Modern hexaploid wheat (AABBDD) is the result ofa more recent hybridization event that occurred *8 000–10 000 years ago and involved an early domesticated tetra-ploid and an ancestral diploid, Aegilops tauschii (DD ge-nome) (Kimber and Feldman 1987; Feldman et al. 1995).The increase in chromosome number and consequent in-crease in genome size poses a challenge to wheat genomics

not experienced with other diploid cereal crops, especially inthe development of high-resolution physical maps based onbacterial artificial chromosomes (BACs) and the assemblyof BAC contigs into their respective sub-genomes. Addition-ally, the presence of 3 related genomes (A, B, and D) addsto the complexity of marker development and analysis, par-ticularly with respect to expressed sequence tags (ESTs),random fragment length polymorphism (RFLP), and simplesequence repeats (SSRs). The prevalence, structure, and in-sertion patterns of repetitive DNA in the wheat genome sug-gest their potential in the development of genome-specificmarkers in wheat physical and genetic mapping (Devos etal. 2005; Paux et al. 2006).

To date, several marker systems based upon TE sequenceshave been developed, mostly based upon polymorphism attheir sites of insertion (Waugh et al. 1997; Ellis et al. 1998;Kalendar et al. 1999). This property has been exploited indeveloping molecular marker systems for genetic analysiswithin cereal grass and legume species (Waugh et al. 1997;Kalendar et al. 1999; Yu and Wise 2000). The sequence-specific amplification polymorphism (S-SAP) markers arethe most popular TE-based markers developed and deployedin barley (Waugh et al. 1997), pea (Ellis et al. 1998), maize(Casa et al. 2000), oat (Yu and Wise 2000), and Medicagosativa (Porceddu et al. 2002). S-SAP is a multiplex ampli-fied fragment length polymorphism (AFLP) technique thatdisplays individual retrotransposon insertions as bands on asequencing gel (Waugh et al. 1997; Ellis et al. 1998). OtherTE-based marker systems include the retrotransposon micro-satellite amplified polymorphism (REMAP) method, whichdetects polymorphisms between amplification of retrotrans-posons proximal to a simple sequence repeat (microsatellite)(Kalendar et al. 1999). In addition, a PCR-based system thatdetects retrotransposon insertion using a primer derived fromthe TE and its flanking DNA sequence has been reported.This system utilizes retrotransposon-based insertion poly-morphism (RBIP), where different allelic states (presenceand absence of the retrotransposon insertion) at a locus canbe revealed (Flavell et al. 1998). Previous studies by Devoset al. (2005) demonstrated the potential of utilizing repeatelement boundaries to generate molecular markers. In thiswork, the authors exploited the RBIP technique to generaterepeat DNA junction markers (RJM) for mapping 4 ran-domly chosen BAC clones to wheat chromosome arms. Re-cently Paux et al. (2006, 2008) and McNeil et al. (2008)demonstrated that BAC end sequences (BES) could be usedto generate insertion-site-based polymorphism (ISBP)markers for genetic and physical mapping in wheat chromo-some 3B. While the work of Devos et al. (2005) and Paux etal. (2006, 2008) demonstrated the use of repeat DNA junc-tions from wheat BACs or BES, the studies did not addressthe use of diploid progenitors to facilitate RJM developmentin hexaploid wheat. In this article we report the rapid devel-opment of genome-specific PCR-based RJM using BES andrandom shotgun sequences from the D genome donor Ae.tauschii for use in hexaploid wheat.

Materials and methods

BAC end sequencingAegilops tauschii BAC clones for BAC end sequencing

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were cultivated in 96-deep-well blocks at 37 8C with shak-ing at 300 r/min for 21 h in 2� YT bacterial growth me-dium supplemented with 1% (v/v) chloramphenicol(Teknova, Hollister, California). Cells were harvested bycentrifugation and BAC DNA preparation was done usingthe R.E.A.L. Prep 96 Plasmid Kit (QIAGEN, Valencia, Cal-ifornia). BAC end sequencing and analysis was carried outas described previously by Huo et al. (2006) but using3.0 mL of BAC DNA in a 10 mL sequencing reaction withthe Big Dye Terminator v3.1 Cycle Sequencing Kit (AppliedBiosystems, Foster City, California).

Primer design from repeat DNA junction regionsRepeat DNA junction primers were designed from *1 600

BES generated in this study, and 5 000 Ae. tauschii–derivedshotgun sequences (Li et al. 2004) downloaded from Gen-Bank. A custom Perl script (http://www.perl.com) RepeatDNA Junction Marker Finder (RJMfinder) program was de-veloped to identify repeat DNA junctions by BLASTN com-paring the Ae. tauschii sequences against the Triticeae repeatsequence database (TREP) (http://wheat.pw.usda.gov/ggpages/ITMI/Repeats/blastrepeats3.html). To maximizeRJM development, BLAST results were also manually ana-lyzed to identify repeat DNA junctions that were not detectedby RJMfinder. The primer spanning the repeat DNA junctionwas designed to have *16 bp of the 5’ sequence upstreamfrom the junction site, and *4 bp downstream from the junc-tion site, independent of the strand. Repeat junction primersdesigned in this automated fashion were manually inspectedto validate the repeat element junctions by blasting the BESand random sequences against TREP (Fig. 1). Primers weredesigned so that one primer in the primer pair spanned therepeat DNA junction while the other primer was designed toamplify a product of approximately 150 to 450 bp (Fig. 2).All primer pairs designed were tested using PCR to evaluate

their amplification success rate on both Ae. tauschii and hex-aploid wheat (Chinese Spring) genomic DNA. Successfulprimer pairs were mapped using nullisomic-tetrasomic (NT)(Sears 1954) and deletion lines (Endo and Gill 1996).

Cytogenetic stocksA set of NT lines from the wheat D genome (N1AT1D,

N2DT2A, N3DT3B, N4DT4A, N5DT5B, N6DT6A, andN7DT7A) (Sears 1954) was used to assign each repeat junc-tion primer pair to individual D-genome chromosomes ofhexaploid wheat. For chromosome arm sub-localization, de-letion lines with breakpoints and bins at various regions ofchromosome 6D were used (Endo and Gill 1996). Repeatjunction primer pairs were mapped to chromosome 6D binsflanked by breakpoints (noted in terms of fraction length,FL) of the largest deletion possessing the marker and thesmallest deletion lacking it. All the aneuploids and deletionstocks were developed in the Chinese Spring genetic back-ground. The genetic stocks were provided by the Wheat Ge-netics Resource Center, Kansas State University, Manhattan,Kansas.

Deletion mappingGenomic DNA from NT lines and controls (Ae. tauschii

and Chinese Spring) was extracted according to the methodreported previously (Riede and Anderson 1996). PCR wasthen performed in a total volume of 20 mL with 5 mmol/Lof each dNTP, 5 mmol/L of each primer, 0.2 units GoTaqpolymerase (Promega, Madison, Wisconsin) with 5� buffer,and 100 ng template DNA. The temperature regime con-sisted of a 4 min initial denaturation step at 94 8C, followedby 35 cycles of 94 8C for 20 s, 57 8C for 20 s, and 72 8C for60 s, and a final extension at 72 8C for 5 min. PCR productswere then separated on 2.5% Metaphor agarose 1� TAEgels (Cambrex Bio-Science Inc., Rockland, Maine).

Fig. 1. BLAST results showing a DNA/RNA type of repeat DNA junction. The junction between a CACTA element (DNA transposon) andan LTR retrotransposon (RNA transposon) is indicated by an arrow.

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Results

Strategy for developing repeat DNA junction markersPreviously, we reported that sequence organization at the

Glu-1 loci is generally conserved between homologouswheat genomes and that the majority of TEs in the inter-genic regions are shared or collinear based on identical in-sertion patterns (Gu et al. 2006). In contrast, TE insertionand sequence organization in the intergenic regions do notappear to be shared between homoeologous A, B, and D ge-nomes, largely due to differential insertion and fast turnoverof TEs after divergence of the A, B, and D genomes (Gu etal. 2004). In the present study, we utilized the sequence con-servation between homologous D genomes (T. aestivum andAe. tauschii) and divergence among homoeologous genomes(A, B, and D of T. aestivum) to generate genome-specificrepeat DNA junction markers for use in hexaploid wheat.This is based on the assumption that the sequence in thejunction region that specifies the insertion of a TE will beunique for the genome carrying that insertion. The first re-quirement for this approach was identification of repeatDNA junctions (Fig. 1) in sequences derived from the D ge-nome diploid progenitor Ae. tauschii using RJMfinder ormanual BLAST. Because transposable elements have de-fined structures with conserved terminal inverted repeat(TIR) motifs, RJMfinder identifies junction regions by thesemotifs. For example, a full-length LTR retrotransposon pos-

sesses either the 5’-TG-3’ or 5’-CA-3’ dinucleotide motif inthe beginning or ending of the LTR sequence. These unique5’ or 3’ TIR motifs were used in the RJMfinder program todetermine the junction of the insertion of a full-length LTRretrotransposon. Similarly, the TIR motif 5’-CACTA-3’ or5’-TAGTG-3’ was used to identify the insertion of CACTAelements, and 5’-TACT-3’ or 5’-AGTA-3’ was used to iden-tify the insertion of MITEs. In the wheat genome, many TEsare truncated or fragmented owing to genomic changes suchas deletion. They often lack recognizable TIR motifs andcannot be detected by RJMfinder. Therefore, manual proc-essing of the BLASTN results was necessary to maximizethe identification of these repeat DNA junction regions.

We screened about 1 600 BES and 5 000 random sequen-ces derived from the D genome donor Ae. tauschii for repeatelement junctions using either automated (RJMfinder Perlscript) or manual BLAST searches against TREP. The pre-cise location of the identified repeat DNA junction was vali-dated by BLAST alignment on TREP between the diploid-derived sequences and the identified repeats. Our analysis ofBES and random sequences from Ae. tauschii revealed thatdifferent types of repeat DNA junctions are present at differ-ent frequencies in the wheat D genome (Table 1). Of the var-ious repeat DNA junctions retrieved from screening Ae.tauschii sequences, 300 were randomly selected to designprimer pairs for further analysis (Tables 1 and 2, Table S12).It was found that 147 (49%) of the repeat DNA junctions

Fig. 2. Strategy for designing repetitive DNA junction primers. The forward primer (F1) in the F1 and R1 primer pair set and the reverseprimer (R2) in the F2 and R2 primer pair set are specific and span the repeat DNA junction. The other primer in each primer pair is fartherfrom the repeat DNA junction, depending on the repeat junction primer parameters and the expected amplification fragment size. Therefore,two primer pair sets can be developed for each repeat junction region. However, only one primer pair set was used for RJM development.

Table 1. Evaluation of success rate of repeat DNA junction primers on Ae. tauschii and hexaploid wheat using PCR.

No. of primers resulting insuccessful amplification

Repeat element junctionNo. of primersdesigned

Hexaploid wheatand Ae. tauschii

Ae. tauschiionly

No. of primers that failed to amplifyAe. tauschii or hexaploid wheat

Retrotransposon–retrotransposon 147 134 3 10Retrotransposon–DNA transposon 35 29 2 4Retrotransposon–other 36 30 0 6DNA transposon–DNA transposon 12 11 0 1DNA transposon–other 70 56 4 10Total 300 260 9 31

Note: Different types of repeat DNA junctions were used to generate primers. Primers derived from Ae. tauschii were analyzed for rate of successfulamplification using PCR on Ae. tauschii and hexaploid wheat genomic DNA.

2 Supplementary data for this article are available on the journal Web site (http://genome.nrc.ca) or may be purchased from the Depositoryof Unpublished Data, Document Delivery, CISTI, National Research Council Canada, Building M-55, 1200 Montreal Road, Ottawa, ONK1A 0R6, Canada. DUD 3945. For more information on obtaining material refer to http://cisti-icist.nrc-cnrc.gc.ca/eng/ibp/cisti/collection/unpublished-data.html.

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were derived from retrotransposon within retrotransposon in-sertions, consistent with the notion that retrotransposons con-stitute a major portion of the wheat genome (SanMiguel etal. 2002; Wicker et al. 2003a, 2003b; Gu et al. 2004). Retro-transposons were frequently associated with each other,forming large blocks of complex nested structures in the in-tergenic regions (SanMiguel and Bennetzen 1998). In con-trast, DNA transposon–derived junctions occurred lessfrequently than retrotransposon-derived junctions. Most ofthe DNA transposons were associated with other non-repetitive sequences (low-copy, coding, or unknown sequen-ces) (Table 1, Table S1).

Analysis of repeat DNA junction markers in diploid andhexaploid wheat

To determine the success rate of this methodology, RJMprimers were tested for amplification using Ae. tauschii ge-nomic DNA. Because TE insertion sites are unique, a primerspanning a specific TE junction typically yields a single am-plification band as visualized on the agarose gel (Fig. 3).Moreover, because one primer of the pair is specific to theunique repeat junction in the genome, the other primer canbe designed either upstream or downstream, irrespective ofthe region’s repetitive or single-copy status (Fig. 2). Of the300 RJM primer pairs tested, 269 (90%) successfully ampli-fied a single fragment from Ae. tauschii genomic DNA, sug-gesting the uniqueness of RJM primers in the genome. Only31 primer pairs (10%) failed or had inexplicable amplifica-tion results (Table 1, Fig. 3).

The RJM primers designed from the Ae. tauschii sequen-ces were used to examine repeat DNA junction conservationbetween the two homologous wheat D genomes. Two hun-dred and sixty nine RJM primer pairs that successfully am-plified Ae. tauschii were tested on hexaploid wheat genomicDNA (Fig. 3; Table 1). Of those 269 RJM primer pairs, 260(97%) produced a single amplification band as visualized onthe agarose gel using hexaploid wheat genomic DNA, while9 (3%) amplified Ae. tauschii genomic DNA only (Fig. 3,lane 2). RJM primers designed from retrotransposons (classI) had a slightly higher rate of successful amplification (198primers or 91%) compared with those designed from DNAtransposons (class II) (71 primers or 86%) (Table 1).

Anchoring repeat DNA junction markers onto wheatchromosomes

Physical mapping of DNA markers in wheat has been fa-cilitated by the availability of a vast wealth of aneuploidstocks (Sears 1954; Endo and Gill 1996). The use of NTlines, where the lack of one chromosome pair is compen-sated by the presence of an extra homoeologous chromo-some, has facilitated the mapping of molecular markers tochromosomes and chromosome arms.

We first anchored the Ae. tauschii–derived RJM onto in-dividual hexaploid wheat D-genome chromosomes using theNT lines of Chinese Spring (Table 2, Fig. 4). Of the 260RJM primer pairs that successfully amplified both diploidand hexaploid wheat, 228 mapped unambiguously to a sin-gle locus on the homologous D-genome chromosomes ofhexaploid wheat, while 32 (12%) produced a single PCRamplification band in all NT lines (Table 2, Fig. 4). There-fore, these 32 primer pairs cannot be unambiguouslyT

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mapped to specific chromosomes of the hexaploid wheat Dgenome. Based on these data, the effective transfer of RJMfrom the diploid D genome to specific chromosomes of hex-aploid wheat is 85% (228/269). Among the 32 RJM primersthat mapped to all NT lines, 16 were from retrotransposon-derived junctions, 14 were from DNA transposon–derivedjunctions, and 2 were from retrotransposon–DNA transposonjunctions (Table 2). Although it is not clear why these pri-mers failed to map to an individual chromosome, the resultcould be partially explained by the observation that a smallnumber of TE insertions are shared between homoeologouswheat genomes (Gu et al. 2004).

Deletion bin mappingWheat aneuploid stocks, including deletion lines, are val-

uable resources for mapping large numbers of wheat EST ormolecular markers to chromosomal bin regions delineatedby neighboring deletion breakpoints (Qi et al. 2003). Toevaluate whether the RJM primers can be precisely anchored

to chromosomal bins on individual chromosomes, the 38 pri-mer pairs assigned to chromosome 6D were further exam-ined by deletion bin mapping. We examined the absence orpresence of expected PCR products in deletion lines missingportions of the chromosome 6D arms (Table 2; Fig. 5). Thisallowed us to locate the mapped primer pairs to their respec-tive sub-chromosomal regions (bins) on the hexaploid wheatchromosome 6D map (Fig. 6). The available deletion binlines divided chromosome 6D into 12 bins. The distributionof deletion bins along chromosome 6D is uneven: 6DL has ahigher number of deletion bins (8 bins) than 6DS (4 bins)(Fig. 6). Of the 38 RJM primer pairs mapped to chromo-some 6D, 31 (82%) were successfully assigned to variousbins on 6D (Fig. 6). The 7 unassigned RJM most likely fallwithin regions of chromosome 6D lacking a deletion bin, es-pecially the centromeric region, which is usually not cov-ered by deletion bin mapping (Qi et al. 2003, 2004).Therefore, these RJM could be useful for mapping at thecentromeric region. On 6DS, 6 markers were located in bin

Fig. 3. PCR amplification of RJM primers tested on diploid Ae. tauschii and hexaploid wheat genomic DNA. Presence of a PCR product onboth hexaploid wheat (Triticum aestivum L. ‘Chinese Spring’) and Ae. tauschii shows that the repeat element junction is conserved on bothD genomes.

Fig. 4. Cytogenetic mapping of RJM primers onto specific hexaploid wheat D-genome chromosomes using Chinese Spring nullisomic-tetrasomic lines. The amplification products of primers RJM-HD003G15R and RJM-HD003I17F assign to chromosomes 7D and 6D, re-spectively.

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6DS6, 3 in bin 6DS4, and 4 in bin 6DS2. Most of the RJMprimer pairs were assigned to 6DL bins, and bin 6DL6(0.29–0.47) had the highest number of RJM (Fig. 6).

Anchoring BAC contigs to the physical mapThe tendency of EST markers to map to all 3 homoeolo-

gous wheat genomes makes them inefficient for preciselyanchoring BACs and BAC contigs on the wheat physicaland deletion maps. Here, we sought to demonstrate the po-tential of RJM for anchoring BACs and BAC contigs ontodeletion maps because of their genome specificity. Ifmapped RJM are derived from end sequences of BAC

clones that have been fingerprinted and assembled into con-tigs, we can directly assign the associated contigs to specificchromosomal bins. Two RJM, RJM-HD003I17F and RJM-HD001O21F, were mapped to deletion bins 6DL6 and6DS6, respectively (Figs. 7A, 7B). The two BACs fromwhich these markers were designed were identified in theirFPC (FingerPrinted Contigs) contigs in the wheat D-genomephysical map database (http://wheatdb.ucdavis.edu). RJM-HD003I17F was located on FPC contig 3320 (Fig. 7A).FPC contig 3320 had 3 associated EST markers, BE426301,BE442681, and BE637610. EST marker BE442681 was as-sociated with multiple contigs, while BE637610 andBE426301 were associated only with contig 3320. Addition-ally, BE426301, BE442681, and BE637610 mapped to mul-tiple bins on homoeologous wheat chromosomes(GrainGenes-SQL database; http://wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi). In this study, RJM-HD003I17F was specifically assigned only to 6DL6(Figs. 6, 7A). Therefore, it is likely that contig 3320 mapsto 6DL6. RJM-HD001O21F was located on contig 9613,which was not previously mapped to the wheat genome ow-ing to lack of anchored markers. Because RJM-HD001O21Fwas derived from the BAC clone in this contig, we can nowassign contig 9613 to 6DS6 (Fig. 7B). Taken together, ourresults demonstrate the value of RJM in accurately anchor-ing contigs to the physical and deletion maps.

Discussion

In the present study, we demonstrated the potential of us-ing the ancestral wheat diploid genomes as a resource forgenerating rapid PCR-based genome-specific repeat DNAjunction markers for use in hexaploid wheat. Owing to thehigh sequence conservation in the intergenic regions betweenhomologous wheat genomes (Gu et al. 2006), the conservedcolinearity and identical insertion pattern of repeat elementsidentified in the ancestral D genome of Ae. tauschii can beutilized as an excellent source of genome-specific repeatDNA markers for the D genome in hexaploid wheat.

Fig. 5. Deletion mapping of RJM primers to chromosome 6D deletion bins. Amplification products with primer RJM-HD003I17F are absenton bins N6D, DT6DL, and 6DL6, indicating that this primer originates from the long arm of chromosome 6D on bin 6DL6. Primer RJM-HD001O21F locates to the short arm of chromosome 6D on bin 6DS6.

Fig. 6. Distribution of RJM along chromosome 6D bins. Numbersin parentheses indicate the bin location, while numbers outside par-entheses indicate the fraction length (FL) of each chromosome bin.RJM allocated to each bin are shown on the right.

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Analysis of genome-specific repeat DNA junctionmarkers

Because of the unique and putatively non-sequence-biasedtransposition mechanism of transposable elements (Ben-netzen 2000), most insertion regions in the genome havebeen observed to be unique. Devos et al. (2005), Paux et al.(2006, 2008), and McNeil et al. (2008) demonstrated the po-tential of using repeat element insertion boundaries to gener-ate molecular markers in hexaploid wheat. These repeatboundary markers were derived from sequences of the hexa-ploid wheat genome. In our study, primers were derivedfrom the diploid D genome, making it theoretically unneces-sary to verify the genome specificity of the markers whenused in hexaploid wheat. We found that only 3% of markersderived from Ae. tauschii failed to map onto the D genomeof hexaploid Chinese Spring, suggesting that approximately97% of repeat junctions are conserved between the two ho-mologous genomes.

The successful use of RJM primers as markers in hexa-ploid wheat depends on conservation of the TE junction be-tween the collinear regions of two homologous genomes.Generally, the Ae. tauschii and T. aestivum D genomes arehighly related and their chromosomes show complete pair-ing (Gill and Raupp 1987). Additionally, previous studieshave demonstrated that most AFLP and RFLP markers areshared and collinear between the Ae. tauschii and T. aesti-vum genetic maps (Boyko et al. 1999). In this work, a trans-ferability of 97% of RJM primers from diploid to hexaploidwheat indicates a very high level of sequence conservationin the flanking regions of the repeat DNA junctions betweenhexaploid wheat and the D-genome progenitor accession(Ae. tauschii) selected in this study. Our result indicates thatthe majority of TEs in hexaploid wheat were inherited fromthe ancestral genomes and that TE insertion patterns andconservation in related homologues have not drasticallychanged since polyploidization.

Accurate identification of repeat junction regions is crit-ical for efficient development of RJM. Our screening of dip-loid wheat sequences revealed a considerable number ofunique repeat DNA junctions: up to 20% of the BES andrandom sequences. The RJMfinder program identified repeatDNA junctions in 11% of the sequences, and we found thatmanual involvement was necessary to identify junctions inan additional 9% of sequences. Paux et al. (2006) reportedthat 10% of BES could be used to develop evenly distrib-uted PCR-based chromosome-specific markers in wheat. Im-provement of the RJMfinder software could result in a betterautomated search result. For example, with the current ver-sion of RJMfinder software, the sequence was blasted onlyagainst TREP. When the TE is inserted into single-copy orgene sequences, manual involvement is necessary to specifythe nature of the repeat junction. Several factors could affectthe results of searches for repeat junctions. In our study, weattempted to include various scenarios of repeat junctions

(Table S1). In addition, the length of each BES and shotgunsequence can directly affect the rate of success in generatingRJM. In general, a shorter sequence will make it harder todesign primers for PCR amplification even if a repeat junc-tion is detected.

Given that retroelements are the major repeat class inplant genomes (Bennetzen 2000), it is not surprising thatretrotransposon-derived junctions provided the highest num-ber of RJM. Retrotransposon-derived RJM also exhibited aslightly higher amplification success rate than DNAtransposon–derived junctions (Table 1). Despite large copynumber, DNA transposons such as MITEs show a strongpreference for insertion into genic DNA, which could leadto more stable and long-lasting integration. Colinear MITEinsertions were identified in the orthologous Glu-1 regionsfrom the A, B, and D genomes of wheat (Gu et al. 2004).Hence, RJM derived from these junction regions will resultin ambiguous or non-scorable PCR amplification, given thetriplicate nature of the hexaploid genome. We found thatRJM from MITE-derived junction regions have a lower suc-cess rate than those from retrotransposon-derived junctionregions.

Repeat DNA junction markers as a tool in wheatmapping

Molecular markers have important applications in manyaspects of plant research, including genetic and physicalmapping, marker-assisted breeding, and map-based cloning(Landjeva et al. 2007). However, development of molecularmarkers from gene or EST sequences for hexaploid wheat isquite difficult owing to the presence of 3 closely related ho-moeologous chromosomes in the genome. In wheat, mostgenes are present in clusters that occur more frequently inthe distal parts of the homoeologous chromosomes (Gill etal. 1996; Erayman et al. 2004). Therefore, markers derivedfrom gene sequences are usually unevenly distributed alongthe chromosomes. In addition, most eukaryotic genomes areoften organized in gene-rich regions separated by gene-poorregions (Sumner et al. 1993). Sequence analyses of large-insert wheat BAC clones and random shotgun sequencesrevealed that repeat DNA, accounting for over 80% of thegenome, is present in genic and intergenic regions (Wickeret al. 2001, 2003b; SanMiguel et al. 2002; Li et al. 2004;Paux et al. 2006; Gu et al. 2006). Hence, repeat DNA se-quences are dispersed throughout the length of all the wheatchromosomes (Paux et al. 2006). This uniform distributiongives RJM an inherent advantage over markers derivedfrom genes or ESTs. Our study showed that RJM are abun-dant in large, polyploid, and highly repetitive genomes.They are unbiased with respect to chromosomal locationsand can be easily generated for both genic and intergenic re-gions, covering the entire genome. They are PCR-basedmarkers and easy to map with high success rates, as com-pared with Southern hybridization–based mapping. In the fu-

Fig. 7. BAC contigs anchored with RJM and EST markers. (A) Contig 3320 is anchored to the physical map by three EST markers. RJM-HD003I17F accurately anchors contig 3320 on 6DL6 on the physical and deletion maps. (B) Contig 9613 lacks any EST marker and isanchored on 6DS6 on the physical map by the marker RJM-HD001O21F.

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ture, a high-throughput platform can be adopted when RJMprimers are florescently labeled and products are separatedby capillary electrophoresis.

RJM have been demonstrated to be a useful tool in Triti-ceae genomics research (Devos et al. 2005; Paux et al. 2006;McNeil et al. 2008). Recently, Paux et al. (2008) furtherdemonstrated the utility of this type of marker for physical,genetic, deletion, and radiation hybrid mapping during theconstruction of a physical map of chromosome 3B of hexa-ploid wheat. In that study, markers were derived mainlyfrom the BES of wheat chromosome 3B–specific librariesconstructed using chromosome-sorting technology (Paux etal. 2006). In the current study, we demonstrated that RJMderived from the ancestral D genome can be directly usedin genome-wide deletion-bin mapping for the D genome ofhexaploid wheat. This will allow us to rapidly develop ge-nome-wide, genome-specific RJM, with no need for furthergenome assignments. This technology could be applied toother wheat genomes, since the other ancestral diploid ge-nomes of hexaploid wheat are well characterized, such asthe A genome of the diploid T. urartu. However, the transferefficiency of RJM from the diploid A genome to the A ge-nome of hexaploid wheat will need to be further examined.

Of 38 RJM primer pairs mapped to chromosome 6D, 31(82%) were successfully assigned to 10 bins on 6D. Our re-sult showed unbiased distribution of RJM along 6D as com-pared with the EST-derived deletion bin map (Qi et al.2003, 2004). Although the physical location of the other 7RJM on 6D were not determined, we know that certain re-gions along the chromosome are not covered by deletionbins, especially centromeric regions, where deletions oftencause lethal effects. Therefore, markers close to the centro-meric regions are often difficult to map. Given that 38 RJMwere unambiguously mapped to chromosome 6D and only31 (82%) of these were anchored to the 10 bins, it can beroughly estimated that 18% of chromosome 6D is not cov-ered by the available deletion bin lines. Deletion bin map-ping provides an efficient approach to map markers todelineated bin locations on an individual chromosome (Qiet al. 2003, 2004). However, deletion bin maps have certainlimitations due to low resolution. The average size of wheatdeletion bins using available deletion lines is *2 Mb andthe markers within the bins are not ordered (Qi et al. 2003).A high-resolution radiation hybrid map with *199 kb/breakhas been reported for the wheat 1D chromosome (Kalava-charla et al. 2006). The RJM will be especially powerful inconstruction of radiation hybrid maps (Paux et al. 2008). Ef-forts are underway to utilize these genome-specific RJM tofacilitate construction of a high-resolution radiation hybridmap for the wheat D genome.

High-resolution BAC-based physical maps are invaluableresources for plant research including genome sequencing,comparative genomics, and map-based cloning of importantagronomic traits (Gupta et al. 2008). However, the largesize, polyploidy, and highly repetitive nature of the hexa-ploid wheat genome make it difficult to develop thousandsof unbiased markers. We imagine that genome-wide physi-cal mapping will be much more challenging in hexaploidwheat because of its triplicate genome. Markers derivedfrom EST and gene sequences will often detect multiple ho-moeoloci among chromosome groups. The unique genome-

specific RJM derived from ancestral genomes could providean alternative strategy for resolving this problem by clearlyseparating and anchoring BAC contigs to specific sub-genomes and chromosome regions. Additionally, RJM con-tribute additional reference points on the genome and addto already existing SSR markers (Roder et al. 1998; Pest-sova et al. 2000) and EST markers (Qi et al. 2003, 2004).The use of RJM will be complementary to other efforts todevelop complete physical, genetic, and radiation hybridmaps for crops with large and highly repetitive genomes.

AcknowledgementsWe thank William Belknap for critical reading of the

manuscript. This work was supported by the United StatesDepartment of Agriculture, Agricultural Research ServiceCRIS No. 532502100-011. The custom Perl script used inthis study to generate RJM primers is available upon request.

ReferencesBennetzen, J.L. 2000. Transposable element contributions to plant

gene and genome evolution. Plant Mol. Biol. 42: 251–269.doi:10.1023/A:1006344508454. PMID:10688140.

Blake, N.K., Lehfeldt, B.R., Lavin, M., and Talbert, L.E. 1999.Phylogenetic reconstruction based on low copy DNA sequencedata in an allopolyploid: the B genome of wheat. Genome, 42:351–360. doi:10.1139/gen-42-2-351. PMID:10231966.

Boyko, E.V., Gill, K.S., Mickelson-Young, L., Nasuda, S., Raupp,W.J., Ziegle, J.N., et al. 1999. A high-density genetic linkagemap of Ae. tauschii, the D-genome progenitor of bread wheat.Theor. Appl. Genet. 99: 16–26. doi:10.1007/s001220051204.

Bureau, T.E., and Wessler, S.R. 1994. Stowaway: a new family ofinverted repeat elements associated with the genes of bothmonocotyledonous and dicotyledonous plants. Plant Cell, 6:907–916. doi:10.1105/tpc.6.6.907. PMID:8061524.

Casa, A.M., Brouwer, C., Nagel, A., Wang, L., Zhang, Q., Kreso-vich, S., and Wessler, S.R. 2000. The MITE family Heart-breaker (Hbr): molecular markers in maize. Proc. Natl. Acad.Sci. U.S.A. 97: 10083–10089. doi:10.1073/pnas.97.18.10083.PMID:10963671.

Chantret, N., Salse, J., Sabot, F., Rahman, S., Bellec, A., Laubin,B., et al. 2005. Molecular basis of evolutionary events thatshaped the hardness locus in diploid and polyploid wheat spe-cies (Triticum and Aegilops). Plant Cell, 17: 1033–1045.doi:10.1105/tpc.104.029181. PMID:15749759.

Devos, K.M., Brown, J.K.M., and Bennetzen, J.L. 2002. Genomesize reduction through illegitimate recombination counteractsgenome expansion in Arabidopsis. Genome Res. 12: 1075–1079. doi:10.1101/gr.132102. PMID:12097344.

Devos, K.M., Ma, J., Pontaroli, A.C., Pratt, L.H., and Bennetzen,J.L. 2005. Analysis and mapping of randomly chosen bacterialartificial chromosome clones from hexaploid bread wheat. Proc.Natl. Acad. Sci. U.S.A. 102: 19243–19248. doi:10.1073/pnas.0509473102. PMID:16357197.

Ellis, T.H., Poyser, S.J., Knox, M.R., Vershinin, A.V., and Am-brose, M.J. 1998. Polymorphism of insertion sites of Ty1-copiaclass retrotransposons and its use for linkage and diversity ana-lysis in pea. Mol. Gen. Genet. 260: 9–19. PMID:9829823.

Endo, T.R., and Gill, B.S. 1996. The deletion stocks of commonwheat. J. Hered. 87: 295–307.

Erayman, M., Sandhu, D., Sidhu, D., Dilbirligi, M., Baenziger,P.S., and Gill, K.S. 2004. Demarcating the gene-rich regions of

Wanjugi et al. 585

Published by NRC Research Press

the wheat genome. Nucleic Acids Res. 32: 3546–3565. doi:10.1093/nar/gkh639. PMID:15240829.

Feldman, M., Lupton, F.G.H., and Miller, T.E. 1995. Wheats. InEvolution of crops. 2nd ed. Edited by J. Smartt and N.W. Sim-monds. Longman Scientific, London. pp. 184–192.

Flavell, A.J., Knox, M.R., Pearce, S.R., and Ellis, T.H.N. 1998.Retrotransposon-based insertion polymorphisms (RBIP) for highthroughput marker analysis. Plant J. 16: 643–650. doi:10.1046/j.1365-313x.1998.00334.x. PMID:10036780.

Gill, B.S., and Raupp, W.J. 1987. Direct genetic transfers from Ae.squarrosa L. to hexaploid wheat. Crop Sci. 27: 445–450.

Gill, K.S., Gill, B.S., Endo, T.R., Boyko, E.V. 1996. Identificationand high-density mapping of gene-rich regions in chromosomegroup 5 of wheat. Genetics, 143: 1001–1012. PMID:8725245.

Gu, Y.Q., Coleman-Derr, D., Kong, X., and Anderson, O.D. 2004.Rapid genome evolution revealed by comparative sequence analy-sis of orthologous regions from four Triticeae genomes. Plant Phy-siol. 135: 459–470. doi:10.1104/pp.103.038083. PMID:15122014.

Gu, Y.Q., Salse, J., Coleman-Derr, D., Dupin, A., Crossman, C.,Lazo, G.R., et al. 2006. Types and rates of sequence evolutionat the high-molecular-weight glutenin locus in hexaploid wheatand its ancestral genomes. Genetics, 174: 1493–1504. doi:10.1534/genetics.106.060756. PMID:17028342.

Gupta, P.K., Mir, R.R., Mohan, A., and Kumar, J. 2008. Wheatgenomics: present status and future prospects. Int. J. Plant Geno-mics, 2008: 896451. doi:10.1155/2008/896451.

Huo, N., Gu, Y.Q., Lazo, G.R., Vogel, J.P., Coleman-Derr, D.,Luo, M., et al. 2006. Construction and characterization of twoBAC libraries from Brachypodium distachyon, a new model forgrass genomics. Genome, 49: 1099–1108. doi:10.1139/G06-087.PMID:17110990.

Kalavacharla, V., Hossain, K., Gu, Y., Riera-Lizarazu, O., Vales,M.I., Bhamidimarri, S., et al. 2006. High-resolution radiationhybrid map of wheat chromosome 1D. Genetics, 173: 1089–1099. doi:10.1534/genetics.106.056481. PMID:16624903.

Kalendar, R., Grob, T., Regina, M., Suoniemi, A., and Schulman,A. 1999. IRAP and REMAP: two new retrotransposon-basedDNA fingerprinting techniques. Proc. Natl. Acad. Sci. U.S.A.98: 704–711. doi:10.1007/s001220051124.

Kimber, G., and Feldman, M. 1987. Wild wheat: an introduction.University of Missouri Press, Columbia, Mo.

Kumar, A., and Bennetzen, J.L. 1999. Plant retrotransposons.Annu. Rev. Genet. 33: 479–532. doi:10.1146/annurev.genet.33.1.479. PMID:10690416.

Landjeva, S., Korzun, V., and Borner, A. 2007. Molecular markers:actual and potential contributions to wheat genome characteriza-tion and breeding. Euphytica, 156: 271–296. doi:10.1007/s10681-007-9371-0.

Li, W., Zhang, P., Fellers, J.P., Friebe, B., and Gill, B.S. 2004. Se-quence composition, organization, and evolution of the core Tri-ticeae genome. Plant J. 40: 500–511. doi:10.1111/j.1365-313X.2004.02228.x. PMID:15500466.

McNeil, M.D., Kota, R., Paux, E., Dunn, D., McLean, R., Feuillet,C., et al. 2008. BAC-derived markers for assaying the stem rustresistance gene, Sr2, in wheat breeding programs. Mol. Breed.22: 15–24. doi:10.1007/s11032-007-9152-4.

Paux, E., Roger, D., Badaeva, E., Gay, G., Bernard, M., Sourdille,P., and Feuillet, C. 2006. Characterizing the composition andevolution of homoeologous genomes in hexaploid wheatthrough BAC-end sequencing on chromosome 3B. Plant J. 48:463–474. doi:10.1111/j.1365-313X.2006.02891.x. PMID:17010109.

Paux, E., Sourdille, P., Salse, J., Saintenac, C., Choulet, F., Leroy,P., et al. 2008. A physical map of the 1-gigabase bread wheat

chromosome 3B. Science, 322: 101–104. doi:10.1126/science.1161847. PMID:18832645.

Pereira, A., Cuypers, H., Gierl, A., Schwarz-Sommer, Z., and Sae-dler, H. 1986. Molecular analysis of the En/Spm transposableelement system of Zea mays. EMBO J. 5: 835–841. PMID:15957213.

Pestsova, E., Ganal, M.W., and Roder, M.S. 2000. Isolation andmapping of microsatellite markers specific for the D genome ofbread wheat. Genome, 43: 689–697. doi:10.1139/gen-43-4-689.PMID:10984182.

Porceddu, A., Albertini, E., Barcaccia, G., Marconi, G., Bertoli,F., and Veronesi, F. 2002. Development of S-SAP markersbased on an LTR-like sequence from Medicago sativa L. Mol.Genet. Genomics, 267: 107–114. doi:10.1007/s00438-002-0643-z. PMID:11919721.

Qi, L., Echalier, B., Friebe, B., and Gill, B.S. 2003. Molecularcharacterization of a set of wheat deletion stocks for use in chro-mosome bin mapping of ESTs. Funct. Integr. Genomics, 3: 39–55. doi:10.1007/s10142-002-0063-5. PMID:12590342.

Qi, L.L., Echalier, B., Chao, S., Lazo, G.R., Butler, G.E., Ander-son, O.D., et al. 2004. A chromosome bin map of 16,000 ex-pressed sequence tag loci and distribution of genes among thethree genomes of polyploid wheat. Genetics, 168: 701–712.doi:10.1534/genetics.104.034868. PMID:15514046.

Riede, C.R., and Anderson, J.A. 1996. Linkage of RFLP markers toan aluminum tolerance gene in wheat. Crop Sci. 36: 905–909.

Roder, M.S., Korzun, V., Wendehake, K., Plaschke, J., Tixier,M.H., Leroy, P., and Ganal, M.W. 1998. A microsatellite mapof wheat. Genetics, 149: 2007–2023. PMID:9691054.

SanMiguel, P., and Bennetzen, J.L. 1998. Evidence that a recent in-crease in maize genome size was caused by the massive ampli-fication of intergene retrotransposons. Ann. Bot. (Lond.), 82:37–44. doi:10.1006/anbo.1998.0746.

SanMiguel, P.J., Ramakrishna, W., Bennetzen, J.L., Busso, C.S.,and Dubcovsky, J. 2002. Transposable elements, genes and re-combination in a 215-kb contig from wheat chromosome 5Am.Funct. Integr. Genomics, 2: 70–80. doi:10.1007/s10142-002-0056-4. PMID:12021852.

Sears, E.R. 1954. The aneuploids of common wheat. Bull. 572.University of Missouri Agricultural Experiment Station, Colum-bia, Mo.

Shirasu, K., Schulman, A.H., Lahaye, T., and Schulze-Lefert, P.2000. A contiguous 66-kb barley DNA sequence provides evi-dence for reversible genome expansion. Genome Res. 10: 908–915. doi:10.1101/gr.10.7.908. PMID:10899140.

Sumner, A.T., Torre, J.D.L., and Stuppia, L. 1993. The distributionof genes on chromosomes: a cytological approach. J. Mol. Evol.37: 117–122. doi:10.1007/BF02407346. PMID:8411200.

Waugh, R., McLean, K., Flavell, A.J., Pearce, S.R., Kumar, A.,Thomas, B.B., and Powell, W. 1997. Genetic distribution ofBare-1-like retrotransposable elements in the barley genome re-vealed by sequence-specific amplification polymorphisms (S-SAP). Mol. Genet. Genomics, 253: 687–694. PMID:9079879.

Wessler, S.R., Bureau, T.E., and White, S.E. 1995. LTR-retrotran-sposons and MITEs: important players in the evolution of plantgenomes. Curr. Opin. Genet. Dev. 5: 814–821. doi:10.1016/0959-437X(95)80016-X. PMID:8745082.

Wicker, T., Stein, N., Albar, L., Feuillet, C., Schlagenhauf, E., andKeller, B. 2001. Analysis of a contiguous 211 kb sequence indiploid wheat (T. monococcum L.) reveals multiple mechanismsof genome evolution. Plant J. 26: 307–316. doi:10.1046/j.1365-313X.2001.01028.x. PMID:11439119.

Wicker, T., Guyot, R., Yahiaoui, N., and Keller, B. 2003a. CACTAtransposons in Triticeae. A diverse family of high-copy repeti-

586 Genome Vol. 52, 2009

Published by NRC Research Press

tive elements. Plant Physiol. 132: 52–63. doi:10.1104/pp.102.015743. PMID:12746511.

Wicker, T., Yahiaoui, N., Guyot, R., Schlagenhauf, E., Liu, Z.,Dubcovsky, J., and Keller, B. 2003b. Rapid genome divergenceat orthologous low molecular weight glutenin loci of the A and

Am genomes of wheat. Plant Cell, 15: 1186–1197. doi:10.1105/tpc.011023.

Yu, G.X., and Wise, R.P. 2000. An anchored AFLP- and retrotran-sposon-based map of diploid Avena. Genome, 43: 736–749.doi:10.1139/gen-43-5-736. PMID:11081962.

Wanjugi et al. 587

Published by NRC Research Press