A Centromeric Tandem Repeat Family Originating From a Part of Ty3/gypsy-Retroelement in Wheat and Its Relatives

Corresponding author: Minoru Murata, Okayama University, Kurashiki 710-0046, Japan., mmura{at}rib.okayama-u.ac.jp (E-mail)

Communicating editor: J. A. BIRCHLER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

From a wild diploid species that is a relative of wheat, Aegilops speltoides, a 301-bp repeat containing 16 copies of a CAA microsatellite was isolated. Southern blot and fluorescence in situ hybridization revealed that 250 bp of the sequence is tandemly arrayed at the centromere regions of A- and B-genome chromosomes of common wheat and rye chromosomes. Although the DNA sequence of this 250-bp repeat showed no notable homology in the databases, the flanking or intervening sequences between the repeats showed high homologies (>82%) to two separate sequences of the gag gene and its upstream region in cereba, a Ty3/gypsy-like retroelement of Hordeum vulgare. Since the amino acid sequence deduced from the 250 bp with seven CAAs showed some similarity (53%) to that of the gag gene, we concluded that the 250-bp repeats had also originated from the cereba-like retroelements in diploid wheat such as Ae. speltoides and had formed tandem arrays, whereas the 300-bp repeats were dispersed as a part of cereba-like retroelements. This suggests that some tandem repeats localized at the centromeric regions of cereals and other plant species originated from parts of retrotransposons.

CENTROMERES play an essential role in precise segregation of sister chromatids at mitosis and meiosis. The DNA structure and associated proteins have been extensively studied in yeasts and humans (reviewed by PIDOUX and ALLSHIRE 2000 ). Data on centromeres have been accumulating in higher plants; however, the centromeric DNA constitution is still unclear (reviewed by MURATA 2002 ). In Arabidopsis thaliana, centromeric regions are composed mainly of tandem repeats, called pAL1 or the 180-bp family (MURATA et al. 1994 ; BRANDES et al. 1997 ; ARABIDOPSIS GENOME INITIATIVE 2000), but retrotransposons and middle repetitive sequences have also been identified (BRANDES et al. 1997 ; COPENHAVER et al. 1999 ). In cereals, two repetitive DNA sequences, CCS1 (ARAGON-ALCAIDE et al. 1996 ) and pSau3A9 (JIANG et al. 1996 ), were first found to localize preferentially at the centromeric regions and were shown later to have similarities to parts of Ty3/gypsy-type retrotransposons (MILLER et al. 1998 ; PRESTING et al. 1998 ). Large-scale sequencing analyses of cosmid, bacterial artificial chromosome (BAC), and/or yeast artificial chromosome (YAC) clones revealed that multiple copies of partial or whole Ty3/gypsy-type retrotranposons are dispersed in the centromeric regions of barley (HUDAKOVA et al. 2001 ), wheat (FUKUI et al. 2001 ), and rice (NONOMURA and KURATA 2001 ). Meanwhile, tandem repeat sequences with <1 kb unit have also been found in the centromeric DNA of common wheat (KISHII et al. 2001 ), maize (ANANIEV et al. 1998 ), pearl millet (KAMM et al. 1994 ), rice (DONG et al. 1998 ), and sugar cane (NAGAKI et al. 1998 ). Contrary to the centromeric retrotransposons, the DNA sequences of which are conserved, these tandem repeats are not conserved even within the same species (ANANIEV et al. 1998 ; CHEN et al. 2000 ; BRENT et al. 2001 ; KISHII et al. 2001 ). However, it was recently shown that 65 kb to 2 Mb clusters of a tandem repeat family (Cent0) with a 155-bp unit are located within the function domains of all rice centromeres (CHENG et al. 2002 ). This suggests that centromeric tandem repeats confer centromere functions in cereals as indicated in A. thaliana and other species.

In this work, we identified a novel tandem repeat family with a unit size of 250 bp in Aegilops speltoides and aimed to characterize its hybridization in the centromeric regions of A- and B-genome chromosomes of wheat and rye chromosomes as well as those of Ae. speltoides chromosomes. The origin and amplification mechanisms of this centromeric repeat family are also discussed.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant materials:
Two lines of Ae. speltoides var. typica (accession nos. KU5727 and KT115-1), kindly provided by Drs. T. R. Endo (Kyoto University) and K. Tsunewaki (Fukui Prefectural University), respectively, were used as genomic in situ hybridization (GISH) probes and a source of repetitive DNA sequences. In addition, the following materials were also used: the octoploid triticale line Y4683 (CHENG and MURATA 2002 ), Secale cereale "Petkus," Triticum monococcum var. vulgare (KT3-1), Ae. squarrosa var. strangulata (KT120-5), T. durum var. Reichenvachii, T. aestivum "Chinese Spring" (CS), Oryza sativa "Nipponbare," Hordeum vulgare "Ishukushirazu," and Avena sativa "Albion."

DNA extraction, cloning, and sequencing:
All genomic DNA were extracted from 1- to 2-week-old seedlings by the Nucleon Phytopure DNA extraction kit (Amersham Bioscience, Arlington Heights, IL). One microgram of Ae. speltoides (KT115-1) DNA was partially digested with 0.02 unit of restriction enzyme Sau3AI at 37° to obtain fragments of 2 kb in length, ligated to pBluescript II SK(+) (Stratagene, La Jolla, CA) linearized with BamHI, and transformed to Escherichia coli XL10-Gold (Stratagene). Colonies were transferred to nylon membranes (Hybond N⁺; Amersham Bioscience) and hybridized with digoxigenin (DIG)-labeled genomic DNA of CS to identify repetitive DNA sequences. The selected clones were sequenced and investigated by fluorescence in situ hybridization (FISH) to find their chromosomal locations.

PCR amplification and sequence data analysis:
The centromeric repeat sequences were obtained by PCR performed with various kinds of genomic DNA as a template, under the conditions of 94° for 30 sec, 59° for 30 sec, and 72° for 30 sec, for 30 cycles. Then the product was purified with SUPREC PCR (Takara Biomedical, Berkeley, CA), ligated to pGEM T-easy vector (Promega, Madison, WI), transformed to E. coli XL10-Gold, and sequenced. The primers used were ZCF1 (5'-CTGGCCTTGAGAAGACGTTC-3'), ZCF3 (5'-CGTTCGAAACAAAGCTCGAT-3'), ZCF4 (5'-GATTATGCGGGAGATTACGAGG-3'), ZCF7 (5'-TCAGCAAATGATGTCTGACCA-3'), ZCR1 (5'-CCTCGTAATCTCCCGCATAA-3'), ZCR2 (5'-GAACGTCTTCTCAAGGCCAG-3'), and ZCR7 (5'-GATGGTCATAATCCCGTACCTG-3'). All sequence data were analyzed by SeqEd version 1.0.3 (ABI, Columbia, MD) and GENETYX-MAC-ATSQ-3.1 (Software Development) software for data processing and GENETYX-MAC 10.1 (Software Development) for homology comparison. We searched the nonredundant nucleic acid sequence database of NR-NT with BLASTN and BLASTX for all sequences. The nucleotide sequences reported here have been registered at DDBJ/EMBL/GenBank under accession nos. AB088401, AB088402 and AB099945, AB099946, AB099947, AB099948, AB099949.

Genomic Southern blot hybridization:
Three micrograms of genomic DNA was digested, electrophoresed and transferred to nylon membranes (Hybond N; Amersham Bioscience), hybridized in hybridization buffer (DIG Easy Hyb; Roche, Indianapolis) with DIG-labeled probes (Roche), and detected by chemical luminescent signals according to the manufacturer's instructions.

Chromosome preparation and FISH:
Germinating seeds were placed at 4° for 24 hr to synchronize cell divisions and transferred to room temperature (RT; 25°). After 24 hr, root tips were collected at the root length of 1.5–2.0 cm and pretreated in ice-cold water for 24 hr. Then they were fixed in ethanol-acetic acid (3:1) and stored at -20°. Chromosome preparations for FISH and GISH were made according to the air-drying technique by MURATA et al. 1992 .

The clones isolated in this study and clone pSc74 (BEDBROOK et al. 1980 ), genomic DNAs of rye, T. monococcum, Ae. squarrosa, and CS were labeled with biotin-14-dUTP (GIBCO BRL, Gaithersburg, MD) or DIG-11-dUTP (Roche) by nick translation. FISH and GISH were carried out using the procedure of MURATA et al. 1992 , MURATA et al. 1997 with minor modifications. In brief, 20 µl of hybridization mixture consisting of 50% (v/v) deionized formamide, 10% (v/v) dextran sulfate, 2x SSC (0.3 M NaCl, 0.03 M sodium citrate), and one or two labeled probes (40 ng each) was used. Avidin-FITC (Roche), streptavidin-Cy3 (Sigma, St. Louis), anti-DIG-fluoroscein (Roche), and/or anti-DIG-rhodamine (Roche) were applied to detect hybridized probes. Fluorescent images were obtained using a fluorescence microscope (Axioskop; Carl Zeiss, Thornwood, NY) with UV-, B-, and triple-band pass filters (Carl Zeiss filter nos. 01, 09, and 25, respectively) and a color-chilled CCD camera (C5810; Hamamatsu Photonics, Bridgewater, NJ). The images were processed using Photoshop 6.0 (Adobe).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Identification and characterization of centromeric repeats from Ae. speltoides:
Genomic DNAs isolated from two lines of Ae. speltoides were labeled with DIG and hybridized together with biotin-labeled rye-specific pSc74 to mitotic metaphase chromosomes of octoploid triticale Y4683. One of the genomic DNA probes (KT115-1) showed a clear banding pattern (Fig 1A). Fourteen chromosomes of common wheat, identified as B-genome chromosomes, showed the strongest hybridization, and distinct signals appeared at the centromeric regions of rye and some other wheat chromosomes. The rye chromosomes were identified by the green telomeric signals from pSc74 probe.

View larger version (111K): In this window In a new window Download PPT slide   Figure 1. GISH and FISH analysis showing localization of the repetitive DNA sequences from Ae. speltoides. (A) Mitotic chromosomes of octoploid triticale (Y4683) hybridized with biotinylated Ae. speltoides genomic DNA and DIG-labeled pSc74, detected with streptavidin-Cy3 (red) and anti-digoxigenin-fluorescein (green), respectively. (B–D) Mitotic chromosomes of common wheat CS counterstained with propidium iodide (B), durum wheat (C), and rye (D), respectively. All were hybridized with DIG-labeled CS2 clone and detected by anti-DIG-fluorescein. Bar, 10 µm.

To isolate the possible repetitive DNA sequences that hybridized preferentially to the centromeric regions, we constructed a Sau3AI partial-digested genomic DNA library from Ae. speltoides KT115-1 and screened it with a DIG-labeled CS genomic DNA probe. After stringent washing (0.1x SSC, 68°), 18 out of 500 clones showed relatively strong signals. All 18 clones were checked subsequently by FISH, and only clone 307-5 showed a FISH pattern similar to the previous GISH pattern (data not shown). The insert was 1083 bp in length and was divided into two parts: 782 bp and 301 bp by a Sau3AI site. Sixteen copies of CAA microsatellite were found in the latter part (Fig 2).

View larger version (60K): In this window In a new window Download PPT slide   Figure 2. The sequence of clone 307-5 (DDBJ/EMBL/GenBank accession no. AB088401). Positions of Sau3AI and SacI sites are boxed. A pair of primers (ZCF1 and ZCR1) and a microsatellite array (CAA)16 are arrowed and underlined, respectively.

Genomic Southern hybridization with clone 307-5 as a probe showed a ladder pattern with an 250-bp repeat unit in the total DNA of Ae. speltoides KT115-1 digested with Sau3AI (data not shown). This suggested that only a part of the insert is arrayed in tandem. To find the tandem sequence, the clone 307-5 DNA was digested with SacI, which cut once at the 684-bp site (Fig 2), self ligated, and transformed. FISH with the resultant clone pBs301 as a probe, which contained a 301-bp Sau3AI fragment from the 3'-end of 307-5, showed a pattern similar to that of 307-5 (data not shown). pBs301 was also used as a probe in Southern hybridization to Sau3AI-digested DNA of CS, durum wheat, and five other diploid species (Fig 3). All species except Ae. squarrosa showed ladder patterns with 250-bp repeat units. S. cereale had slightly faint 250-bp bands, whereas T. monococcum had a strong 500-bp band corresponding to a 250-bp-unit dimer. Both lines of Ae. speltoides, especially KU5725, showed a heavy smear, but the ladder patterns could be observed when the exposure time was short.

View larger version (66K): In this window In a new window Download PPT slide   Figure 3. Southern blot hybridizations of Sau3AI-digested genomic DNA of nine cereal species with pBs301 as a probe.

The smear observed in Ae. speltoides lanes was probably produced by the microsatellites of (CAA)₁₆ in the pBs301 probe, since some microsatellites are known to cause smearing in Southern blotting and dispersed signals in FISH (SCHMIDT and HESLOP-HARRISON 1996 ). This assumption was also supported by the size of a Sau3AI fragment in pBs301, which is 250 bp when the microsatellite repeats (48 bp) are subtracted. To identify the putative 250-bp repeat and remove the effect of the microsatellite, we designed PCR primers of ZCF1 and ZCR1 to amplify the 288-bp fragment (named pBs301-1) of pBs301 (Fig 2). As a result, two bands of 250 and 300 bp in length were amplified in almost all species used (Fig 4A).

View larger version (123K): In this window In a new window Download PPT slide   Figure 4. Agarose electrophoresis of PCR products amplified by the primer combinations of ZCF1 + ZCR1 (A) and ZCR2 + ZCF3 (B). Genomic DNAs used as a template were: (1) CS (T. aestivum), (2) T. durum, (3) Ae. speltoides (KT115-1), (4) Ae. speltoides (KU5727), (5) T. monococcum, (6) Ae. squarrosa, and (7) S. cereale.

To investigate the copy numbers of the microsatellite, we cloned and sequenced some of the PCR products. Two clones from CS (CS2, 245 bp and CS3, 253 bp; Fig 5) and two clones from Ae. speltoides KT115-1 (253 and 247 bp) contained only two to four copies of CAA microsatellite. All of these clones hybridized preferentially to the centromeric regions of 28 chromosomes in common wheat (Fig 1B), 28 in durum wheat (Fig 1C), and 14 in Ae. speltoides (data not shown) or in rye (Fig 1D). In Southern blot hybridization to Sau3AI-digested genomic DNA, both 250-bp monomeric and 500-bp dimeric bands appeared without smearing in common wheat, durum, and Ae. speltoides, and a 500-bp band appeared in T. monococcum (data not shown). Although the lengths of monomers were variable (245–253 bp), the sequences appeared to be tandemly arranged within the centromeric regions in A- and B-genome chromosomes of wheat and R-genome chromosomes of rye. By contrast, no signals were found in rice, barley, millet, or oats (data not shown).

View larger version (87K): In this window In a new window Download PPT slide   Figure 5. DNA sequence alignment of five clones from common wheat and pBs301-1. The conserved nucleotides are in black boxes. The PCR primers used are also indicated.

As shown in a sequence alignment of five PCR clones of CS to pBs301-1 of Ae. speltoides (Fig 5), all clones contained more than one copy of a CAA microsatellite and its derivatives. The most common substitutions were a transversion from A to T, but A G and C T transitions also occurred. Compared with the variation of the microsatellite, the sequences flanking the microsatellites were conserved, although a 35-bp deletion in CS15, a 9-bp deletion in CS2, and short 1- to 4-bp deletions were in all clones. Similarly, in five PCR clones from Ae. speltoides (KT115-1), 3–16 copies of the CAA microsatellite were found. However, 20 nucleotides just upstream the microsatellite arrays were less conserved than those of CS clones. These and other data indicated that 250-bp sequences having a low copy number of CAA microsatellites are arrayed in tandem, but 300-bp sequences, which have more copies, are located separately.

Intervening sequences among the 250- and 300-bp repeats:
To amplify intervening sequences between the 250- and 300-bp repeats, we designed three different PCR primers: ZCF3, ZCF4, and ZCR2 from the conserved sequences among CS and pBs301-1 clones (Fig 5 and Fig 6A). The combination of ZCF3 and ZCR2 amplified >10 bands 450 bp to 5 kb in length from CS DNA (Fig 4B, lane 1), while the primer ZCF4 and ZCR2 combination produced only 2 faint bands. Similar amplification results were also obtained using other genomic DNA as a template (Fig 4B, lanes 2–7). The products from Ae. speltoides (KT115-1), Ae. Squarrosa, and CS were cloned and end sequenced. Out of 19 clones, 16 contained sequences homologous to parts of cereba, a Ty3/gypsy-like retroelement of Hordeum vulgare (HUDAKOVA et al. 2001 ; GenBank accession nos. AY040832 and AF078801; Fig 6A). Two separate regions of the cereba DNA showed high homologies (>82%) to the PCR products amplified from a primer combination of ZCF3 and ZCR2: one is an 80-bp 5'-region of gag gene (nucleotide position 1399–1479 in AY040832) and its upstream noncoding regions [5'-untranslated region (UTR)] in different lengths; another is the coding regions 34–105 bp long in the gag gene, all of which started at position 1926 (Fig 6A). This revealed a distinct nonhomologous sequence 450 bp long between the two homologous regions in the gag gene. Although no homology at the DNA sequence level was found between the sequence 1479–1926 of cereba and the 250- to 300-bp repeat such as pBs301, the amino acid sequence deduced from pBs301 showed 53% similarity (41% identity) to that from the DNA sequence 1531–1824 of cereba (Fig 6B). This suggests that the 250- and 300-bp repeats had originated from Ty3/gypsy-like retrotransposons like cereba in H. vulgare and that there are cereba-like retroelements are in the A and B genomes of wheat, but their sequences corresponding to 1531–1824 in cereba (AY040832) vary much from that of cereba. To confirm this finding, we designed a new pair of PCR primers on the basis of the cereba sequences (GenBank accession no. AF078801), ZCF7 and ZCR7, which are expected to amplify a 458-bp fragment (3292–3749 in cereba, no. AF078801) or a 485-bp fragment (1467–1951 in cereba, no. AY040832; Fig 6A). The primer pair could amplify the expected size fragments from barley, having almost the same sequence as cereba, while all four PCR products from Ae. speltoides (KT115-1) contained the 250- to 300-bp sequences. The DNA sequence of clone ZC7-1 was almost identical to that of pBs301-1 except for the copy number of CAA microsatellite, which was reduced to five.

View larger version (38K): In this window In a new window Download PPT slide   Figure 6. Schematic representations showing the relationship between the 250- and 300-bp repeats and cereba, a Ty3/gypsy-like retroelement of H. vulgare. (A) A part of cereba containing a long terminal repeat (LTR), a primer-binding site (PBS), and a gag gene. Numbers correspond to the nucleotide positions of a cereba (accession no. AY040832). Arrows and arrowheads indicate the regions having high homologies (>82%) to the PCR products amplified by a combination of ZCF3 and ZCR2 primers. (B) An alignment of amino acid sequences deduced from pBs301 and a part of the gag gene (1531–1824 of AY040832). (C) Estimated organization of the 250- and 300-bp repeats and nonhomologous sequences found in the up- and downstream regions of the 250- to 300-bp repeats.

In the PCR products from CS as well as Ae. speltoides, however, we found highly conserved 40- and 48-bp sequences at the junctions of the 250- to 300-bp repeats and cereba-like sequences, showing no homology to that of either cereba or pBs301 (Fig 6C). The origin of the two sequences is uncertain, since no sequences showing overall homology to either 40 or 48 bp were found in the DNA databases.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Ae. speltoides (2n = 2x = 14, genome constitution SS) has been shown to have the highest genetic affinity to the B genome of common wheat (reviewed by DVORAK and ZHANG 1990 ; FRIEBE and GILL 1996 ; TSUNEWAKI 1996 ). In our study, genomic DNA from Ae. speltoides painted B-genome chromosomes more heavily than A-genome chromosomes and produced interstitial and pericentric bands (Fig 1A). This GISH pattern was produced mainly by a 250-bp centromeric repeat and a CAA microsatellite, since the GISH pattern was very similar to the FISH pattern obtained with the pBs301 clone containing both the 250-bp repeat unit and 16 copies of CAA microsatellite as a probe. Furthermore, the 250-bp repeat with a few copies of the microsatellite such as pBs301-1 hybridized only to centromeric regions of A-, B-, and R-genome chromosomes as well as S-genome chromosomes (Fig 1).

Several tandem repeat families have been reported to be present in the centromeric regions of cereal chromosomes (ANANIEV et al. 1998 ; DONG et al. 1998 ; KISHII et al. 2001 ). However, no homology was found among the present 250-bp repeat and other tandem repeats. This poor conservation is contrary to the extensive presence of CCSI and pSau3A9 repeats in cereal species (ARAGON-ALCAIDE et al. 1996 ; JIANG et al. 1996 ). DNA sequences of CCS1 and pSau3AI are similar to those of the LTR and of the integrase gene, respectively, in Ty3/gypsy-like retroelements (MILLER et al. 1998 ; PRESTING et al. 1998 ). Although the DNA sequences of the 250-bp repeats showed no homology to cereba (PRESTING et al. 1998 ; HUDAKOVA et al. 2001 ) or any other retrotransposon-like sequences, their flanking or intervening sequences between the repeats showed high homology (>82%) to two separate sequences of gag gene and its upstream region (5'-UTR) in cereba (Fig 6A). This suggests that a number of cereba-like retroelements exist in the A and B genomes of wheat and that the 250-bp repeats are also related to cereba-like sequences. This suggestion was clearly supported by the 53% similarity obtained between the deduced amino acid sequences from the 250 bp with several CAAs and of the gag gene of cereba (Fig 6B), indicating that the DNA sequences corresponding to the region [nucleotide position 1480–1925 in cereba (GenBank accession no. AY040832)] are highly divergent from barley to wheat.

FUKUI et al. 2001 reported the presence of cereba-like retroelements in wheat centromeric regions, although they did not find the 250-bp tandem repeats or the region nonhomologous to cereba. So it is quite interesting to know whether cereba-like retroelements in wheat, named here crew (centromeric retroelements of wheat), are still active or not. In most of our PCR products from wheat and Ae. speltoides, more than one stop codon appeared when their DNA sequences were translated into amino acids, but a few were uninterrupted. Like cereba in barley (HUDAKOVA et al. 2001 ), therefore, complete and possibly autonomous Ty3/gypsy-like retroelements may be present in the genomes of wheat and/or it relatives.

As shown in Fig 6C, in the centromeric regions of wheat and Ae. speltoides, the 250-bp repeats with a few copies of CAA microsatellite are thought to be arrayed in tandem, but those with many copies are thought to be dispersed. This raises a question: Why is only the 250-bp unit amplified in tandem? Tandem repeats are common in centromeric regions of higher eukaryotes, and a number of amplification mechanisms have been suggested. The amplification of this tandem repeat was probably caused by the abundance of the crew sequences in the centromeric regions. We found highly conserved sequences (40 and 48 bp, respectively; Fig 6C) at both the junctions of the 250- to 300-bp repeats and the sequences homologous to cereba in crew. Since they had no high homology to cereba, pBs301, or any other sequences in the DNA databases, their origin is uncertain. They might be important for amplifying the repeat in tandem, such as functioning as hot spots for recombination. Recombination might occur between a (CCA)₂(GCA)(CCA)(CAA)₂(CCA) microsatellite array located at the end of the 48-bp junction sequence (Fig 6C) and the CAA microsatellite array in the 250- to 300-bp repeats.

Microsatellite variability is proposed to be generated by "DNA replication slippage," and to require a minimum number of repeats (reviewed by SCHLOTTERER 2000 ). As described above, no DNA sequence homology between the region [nucleotide position 1480–1925 in cereba (accession no. AY040832)] and the 250-bp repeat was detected, but the enforced alignment between them revealed that the nucleotide sequence CAACCA(CAA)₂C in cereba (1630–1642 in accession no. AY040832) can be aligned with (CAA)₄C in pBS301-1 (96–108, Fig 5). This indicates that a (CAA)₄C sequence is a prototype of the microsatellite array. Both cereba in barley and crew in Ae. speltoides and wheat presumably originated from a single ancestral retrotransposon family, like crwydryn (LANGDON et al. 2000 ). However, the 250-bp repeat of crew evolved to have many copies of CAA microsatellite within the sequence or popped out to form tandem arrays, while the corresponding sequences in cereba remained stable in the evolutionary process.

Tandem repeats are abundant components of centromere DNA in higher eukaryotes and have been thought to be related to centromere function, although their sequences are not homologous to each other. This study demonstrated the retroelement relationship of a novel centromeric 250-bp tandem repeat family and the possible origin and amplification mechanisms, suggesting that some tandem repeats localized at the centromeric regions of cereals and other plant species originated from parts of retrotransposons.

	ACKNOWLEDGMENTS

We thank Dr. J. S. (Pat) Heslop-Harrison for critical reading of this manuscript. This work was supported by the Core Research for Evolutionary Science and Technology Program and Japan Science Technology Corporation.

Manuscript received October 4, 2002; Accepted for publication February 17, 2003.

	LITERATURE CITED

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