Dasheng: A Recently Amplified Nonautonomous Long Terminal Repeat Element That Is a Major Component of Pericentromeric Regions in Rice

Corresponding author: Susan R. Wessler, University of Georgia, Athens, GA 30602., sue{at}dogwood.botany.uga.edu (E-mail)

Communicating editor: C. S. GASSER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

A new and unusual family of LTR elements, Dasheng, has been discovered in the genome of Oryza sativa following database searches of 100 Mb of rice genomic sequence and 78 Mb of BAC-end sequence information. With all of the cis-elements but none of the coding domains normally associated with retrotransposons (e.g., gag, pol), Dasheng is a novel nonautonomous LTR element with high copy number. Over half of the 1000 Dasheng elements in the rice genome are full length (5.6–8.6 kb), and 60% are estimated to have amplified in the past 500,000 years. Using a modified AFLP technique called transposon display, 215 elements were mapped to all 12 rice chromosomes. Interestingly, more than half of the mapped elements are clustered in the heterochromatic regions around centromeres. The distribution pattern was further confirmed by FISH analysis. Despite clustering in heterochromatin, Dasheng elements are not nested, suggesting their potential value as molecular markers for these marker-poor regions. Taken together, Dasheng is one of the highest-copy-number LTR elements and one of the most recent elements to amplify in the rice genome.

TRANSPOSABLE elements (TEs) have been divided into two classes, class 1 or RNA elements and class 2 or DNA elements. An RNA intermediate and a replicative mechanism of transposition are involved in the transposition of class 1 elements (LEWIN 1997 ). RNA elements can be further divided into several groups, including long terminal repeat (LTR) retrotransposons, long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs). RNA elements are capable of attaining very high copy numbers because the element-encoded mRNA, not the element itself, forms the transposition intermediate.

LTR retrotransposons make up the largest fraction of most plant genomes (KUMAR and BENNETZEN 1999 ). The LTRs usually contain the initiation and termination sites of a transcript that encodes at least two genes, gag and pol. The products of these genes are involved in the different steps of retrotransposition, including reverse transcription and integration (LEWIN 1997 ; Fig 1). Immediately internal to the LTR is the primer binding site (PBS) and the polypurine tract (PPT). Both are important cis-elements that are necessary for the initiation of the synthesis of element DNA from the RNA intermediate. LTR elements are classified into two types on the basis of the order of their encoded genes: Ty1/copia and Ty3/gypsy elements (XIONG and EICKBUSH 1990 ). Both are prevalent in plant genomes (VOYTAS et al. 1992 ; SUONIEMI et al. 1998 ).

View larger version (16K): In this window In a new window Download PPT slide   Figure 1. Comparison of Dasheng with a typical autonomous LTR retrotransposon. Coding regions are shown as hatched boxes. CP, capsid-like proteins; PR, protease; INT, integrase; RT, reverse transcriptase; RH, RNase H. The relative order of RT, RH, and INT varies with different types of elements (see text). Other sequences indicated are: LTR, long terminal repeat; PBS, primer binding site; PPT, polypurine tract; NA, nucleic acid binding moiety. Arrows above the LTR of Dasheng indicate the positions of transposon-specific primers (Pr1 and Pr2) for transposon display. These would be used for PCR with the adapter primer shown (see Fig 2 and text).

Differential amplification of LTR retrotransposons has been shown to be largely responsible for the C-value paradox in members of the grass clade (CHEN et al. 1997 ; SANMIGUEL and BENNETZEN 1998 ; DUBCOVSKY et al. 2001 ). The C-value paradox refers to the lack of correlation between the genome size and the biological complexity of an organism (THOMAS 1971 ). For example, rice (Oryza sativa) and barley (Hordeum vulgare) have roughly the same number of genes and a largely conserved gene order (MOORE et al. 1995 ; DUBCOVSKY et al. 2001 ). The 11-fold difference in the size of their genomes (430 vs. 4800 Mb) is due, in part, to the fact that retrotransposons comprise more than half of the barley genome and only 14% of the rice genome (VICIENT et al. 1999 ; TARCHINI et al. 2000 ). With the International Rice Genome Sequencing Program scheduled for completion in less than a year (MYERS 2001 ), new insights about the identity and frequency of different TE families will emerge.

Despite its small genome, rice is still a model organism for the study of transposable elements. The genome of O. sativa contains all of the major types of elements found in the larger grass genomes, including retrotransposons, miniature inverted repeat transposable elements (MITEs), and other DNA elements (BUREAU et al. 1996 ; MAO et al. 2000 ; TARCHINI et al. 2000 ; TURCOTTE et al. 2001 ). Furthermore, the availability of several well-characterized wild relatives provides the material necessary to analyze the impact of TEs on genome evolution and speciation. O. sativa is composed of two cultivated subspecies (indica and japonica) with thousands of diverse cultivars distributed worldwide. The genus Oryza has >20 species whose evolutionary relationships have been the subject of several phylogenetic analyses (UOZO et al. 1997 ; GE et al. 1999 ; SHARMA et al. 2000 ).

In this study, database searches of 100 Mb of rice genomic sequence and 78 Mb of bacterial artificial chromosome (BAC) end sequence led to the identification of a new and unusual family of LTR elements called Dasheng. Dasheng is a very recently amplified family of 800–1300 nonautonomous elements, making it one of the most recently amplified and highest-copy-number families in rice. The family also includes 16% solo LTRs. Like many other high-copy-number LTR elements, Dasheng elements are concentrated in the gene-poor pericentromeric regions of the chromosomes, which might be the reason for Dasheng's success in the small genome of rice. The availability of large amounts of genomic sequence and an almost completely assembled chromosome 1 has allowed us to address questions regarding the distribution and timing of insertion events and to test models that explain the formation of solo LTRs.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Plant material and DNA extraction:
A doubled haploid (DH) mapping population (GUIDERDONI et al. 1992 ; HUANG and KOCHERT 1994 ) was used in conjunction with an existing SSR mapping data set (TEMNYKH et al. 2001 ) to map Dasheng elements. This population consisted of a subset of 96 doubled haploid lines derived via anther culture from the inter-subspecific cross between IR64 (O. sativa ssp. indica) and Azucena (tropical japonica). Other rice cultivars and wild species were obtained from the McCouch lab (Cornell University) and Gary Kochert (University of Georgia). Plant DNA was extracted as described (MCCOUCH et al. 1988 ).

Genetic mapping:
Transposon display was performed as described in CASA et al. 2000 to generate segregation patterns in the DH population with the following modifications. The element-specific primers were derived from the LTR sequence of Dasheng and the reaction was performed with rice DNAs. The final annealing temperature for selective amplification was 58° with ³³P-labeled Dasheng primer. Sequences of primers are available upon request. DNA fragments from transposon display were excised and cloned as described (CASA et al. 2000 ). DNA templates were sequenced by the Molecular Genetics Instrumentation Facility (University of Georgia).

The gel images of transposon display with DNAs from the DH mapping population were scored manually for presence/absence of polymorphic bands corresponding to Dasheng elements. The Dasheng markers were integrated into the SSR framework map using the Kosambi mapping function and MapMaker 3.0 software (LANDER et al. 1987 ). Markers with a ripple of LOD > 2.0 were integrated into the framework maps and those mapping with LOD < 2.0 were assigned to the most likely intervals.

Fluorescence in situ hybridization analysis:
Fluorescence in situ hybridization (FISH) analysis was performed as previously described (JIANG et al. 1995 ) using Nipponbare and indica cultivar Zhongxian 3037. The Dasheng probe (Fig 1) was labeled with biotin-16-UTP and detected using a fluorescein-5-isothiocyanate (FITC)-conjugated antibiotin antibody (Vector Laboratories, Burlingame, CA). Propidium iodide in an antifade solution was used to counterstain the chromosomes. Chromosome and FISH signal images were captured using a SenSys charge-coupled device camera (Photometrics, Tucson, AZ) and analyzed using IPLab Spectrum software (Signal Analytics, Vienna, VA).

DNA sequence analysis:
DNA sequence analyses (pairwise comparisons, multiple sequence alignments, sequence assembling, and formatting) were performed with programs in the University of Wisconsin Genetics Computer Group program suite (version 10.1) accessed through Research Computing Resources, University of Georgia.

Identification of repetitive sequences from BAC ends:

All sequences in the rice BAC end database (O. sativa cv. Nipponbare) were downloaded from the website of Clemson University Genome Institute (http://www.genome.clemson.edu) for the initial analysis (August 1999). An all vs. all comparison was performed with the sequences using WUBLASTN (http://blast.wustl.edu) with parameters M = 5, N = -11, Q = 22, R = 11, -kap E = 0.001, -hspmax 5000). Groups with highest intragroup similarities (>95%) were further characterized with BLAST search in the National Center for Biotechnology Information (NCBI) server (http://www.ncbi.nlm.nih.gov).

Identifying transposable elements in genomic sequence:

The sequences of rice BACs and PACs were downloaded from the web sites of different rice genome projects including groups in the United States (http://www.usricegenome.org/), Japan (http://rgp.dna.affrc.go.jp/), Korea (http://bioserve.myongji.ac.kr/ricemac.html), People's Republic of China (http://www.ncgr.ac.cn/Ls/index.html), and Taiwan (http://genome.sinica.edu.tw/). Completely sequenced PACs or BACs and those in annotation and finishing (phase 2) were used as query sequences to search for transposable elements with RepeatMasker (A. Smit and P. Green, http://ftp.genome.washington.edu/RM/webrepeatmaskerhelp.html) as described (JIANG and WESSLER 2001 ).

Copy-number determination:
The copy number of Dasheng was estimated in three ways:

1. By blasting BAC ends using LTR sequence as a query. Using this method, copy number = matches in BAC ends x 430 Mb (rice genome size) ÷ the size of the BAC ends database (in megabases).

2. By probing a rice BAC library [derived from Nipponbare (MAO et al. 2000 )] with a 500-bp fragment located between the third tract of direct repeats and the PPT (see Fig 1). Using this method, copy number of Dasheng elements = (number positive clones ÷ number of BACs screened) x 430 Mb ÷ average size of BACs (in megabases). The raw value, estimated to be 700 elements per haploid genome, was corrected for the number of solo LTRs (16%), BACs and PACs containing two or more elements (12% of the positive clones), and truncated elements (30%). The corrected copy number was 900–1300 (depending on the percentage of truncated elements detected).

3. By screening the genomic sequence with RepeatMasker followed by manual examination. The copy number = number of elements in genomic sequences x 430 Mb ÷ total size of the genomic sequence screened.

The copy number of other rice LTR elements (elements reported previously and those identified in this study) was estimated by blasting the BAC end database and GenBank (NCBI BLAST server) with LTR sequences. Low score matches (e > 10^-30) from GenBank were checked manually to determine whether the matches represented the element. The copy number for each element = matches for this element x 430 Mb ÷ total size of the rice genomic sequence in GenBank.

Phylogenetic analysis and aging of elements:
LTR nucleotide sequences homologous to Dasheng and RIRE2 were aligned using GCG (see above). Tree production and bootstrap analyses were performed using PAUP version 4.0. Sequence similarities and standard error were calculated with MEGA program (KUMAR et al. 2001 ). Full-length elements were aged (as in SANMIGUEL et al. 1998 ) by comparing their 5' and 3' LTR sequences. Kimura-2 parameter distances (K) between 5' and 3' LTRs of individual elements were calculated using MEGA. An average substitution rate (r) of 6.5 x 10^-9 substitutions per synonymous site per year for grasses (GAUT et al. 1996 ) was used to calibrate the ages of the elements. The time (T) since element insertion was estimated using the following formula: . Fifty-percent consensus sequences were determined from group-specific alignments using the EMBL consensus sequence server (http://www.bork.embl-heidelberg.de/Alignment/consensus.html).

The distribution of Dasheng elements in genomic sequences:
The distribution of Dasheng on chromosome 1 of Nipponbare was constructed according to the positions of PACs and BACs that contained Dasheng elements (http://rgp.dna.affrc.go.jp/). Estimates of physical:genetic distance and insertion frequency were based on the data provided by the Rice Genome Research Program (http://rgp.dna.affrc.go.jp/) at the time of analysis. DNA density for chromosomal arms and pericentromeric regions was calculated from the total DNA of three contigs (20.2–34.5 cM and 40–50 cM in arms, 60–70 cM in pericentromeric regions) on chromosome 1. The borders of pericentromeric regions were defined as 15 cM from the center of the centromere on each arm. The position of the centromere was according to HARUSHIMA et al. 1998 and CHENG et al. 2001A . The remainder of the chromosome was defined as arms. Physical:genetic distance equals the physical length of DNA in base pairs divided by the map units covered. Insertion frequency equals the number of elements found in a certain region divided by the physical length of DNA in that region. The total amount of DNA was the size of all the clones minus overlap.

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

A nonautonomous LTR element with very high copy number:
To identify repeat sequences that might be novel transposable elements, we performed an all vs. all comparison with BAC end sequences of rice (O. sativa ssp. japonica cv. Nipponbare; see MATERIALS AND METHODS for details). Several groups of BAC ends were distinguished by their high within-group sequence similarity (95%). The sequence of each group was then used as a query to perform further searches in GenBank. Significant matches for all groups were found in an 8.6-kb segment of a PAC clone from rice chromosome 6 (GenBank accession no. AB023482). This region has the structural features of an LTR retroelement including a long terminal repeat (441 bp with 99.5% sequence similarity), an adjacent putative PBS and PPT, and a 5-bp target site duplication flanking the LTR (Fig 1). The 441-bp LTR is related (65–70% sequence similarity) to the LTR of RIRE2, a previously described Ty3/gypsy type LTR element in rice (OHTSUBO et al. 1999 ). In addition, the two elements also have similar PBSs and PPTs that differ only at 1 or 2 out of 15 nucleotides.

Despite having structural features of LTR retrotransposons, the 7.8-kb region between the LTR contains only very short open reading frames with no similarity to known proteins. Instead, 4 kb of this region is composed of tandem repeats of an 89- to 90-bp unit (Fig 1). The other part of the internal region includes a hypervariable domain (of 0–1.2 kb) located between the PBS and the first tract of tandem repeats (Fig 1). The lack of coding capacity suggests that this element is most likely nonautonomous.

The copy number of this element family (named Dasheng) was estimated in three ways (see below and MATERIALS AND METHODS for details). On the basis of the prevalence of the LTR sequence in BAC ends [150 hits in 78 Mb of HindIII- and EcoRI-digested sequences (e 10^-15)], we estimate that 800 copies of Dasheng are in the genome of cv. Nipponbare. To test whether the prevalence of Dasheng in BAC ends is representative of the rest of the genome, a BAC library of the cv. Nipponbare genome was screened with a Dasheng probe. This experiment led to a copy number estimate of 900–1300. In contrast, a search of 100 Mb of publicly available assembled genomic sequence led to a copy number determination of 470 per haploid genome or approximately one element per megabase. The two- to threefold difference in the values obtained from BAC screening and BAC end sequences vs. genomic sequence may be due to the fact that the latter is biased toward gene-rich regions, whereas several LTR retrotransposon families are enriched in pericentromeric regions of the genome (MILLER et al. 1998 ; LANGDON et al. 2000 ; NONOMURA and KURATA 2001 ; also see below).

The copy number of Dasheng was also compared with that of other LTR elements in rice. This was done by querying the BAC end and genomic sequence databases with LTRs from several high-copy-number rice elements previously described and elements identified in this study (see MATERIALS AND METHODS). As with the searches using Dasheng sequences as queries, the results were inconsistent from one database to the other. The average values obtained (from BAC ends and from genomic sequence) were, in descending order of copy number, Retrosat2 (1080; GenBank accession no. AF111709), Bajie (730; identified in this study), RIRE4 (730; KUMEKAWA et al. 1999), SZ-19 (725; identified in this study), Dasheng (635), RIRE8 (620; KUMEKAWA et al. 1999 ), RIRE3 (510; KUMEKAWA et al. 1999 ), RIRE2 (420; OHTSUBO et al. 1999 ), RIRE9 (115; HAN et al. 2000 ; LI et al. 2000 ), and RCS1 (90; DONG et al. 1998 ).

In a prior study, dot blot hybridization led to a copy number determination for the RIRE2 family of 10,000 in IR36 (OHTSUBO et al. 1999 ). In contrast, we found that the number of hits using RIRE2 sequences was no higher than that found for Dasheng. The striking discrepancy may be due to the presence of distantly related families, a frequent cause of copy number overestimation when employing hybridization methods (MEYERS et al. 2001 ).

The chromosomal location of Dasheng elements: Genetic mapping of Dasheng elements:
To determine the chromosomal distribution of Dasheng, family members were mapped using a technique called transposon display, which is a modification of the amplified fragment length polymorphism (AFLP) procedure that generates PCR products anchored in a transposable element and a flanking restriction site (WAUGH et al. 1997 ; VAN DEN BROECK et al. 1998 ; CASA et al. 2000 ). The number of fragments amplified in one reaction can be adjusted by adding extra bases to the adapter primer (so-called selective bases); fewer fragments will be detected with more selective bases (VOS et al. 1995 ). Transposon display has the added advantage of detecting solo LTRs since the transposon-specific primers are located within the LTR (Fig 1, Pr1 and Pr2).

Dasheng primers were designed so as not to recognize the related RIRE2 elements. Insertion site polymorphism, as defined by the presence of a PCR product in one parent but not in the other, was high for the parents (IR64 and Azucena), varying from 76.3 to 84.3% for different adapter primer/enzyme combinations in this inter-subspecific cross (Table 1; Fig 2A). High levels of polymorphism were also detected within indica and japonica subspecies (Fig 2B and Fig C), indicating that Dasheng elements can serve as a valuable marker system. Several wild species of rice were also tested with the same enzyme but they needed more selective bases (MseI + T for O. sativa; MseI + TG for wild species; Fig 2A and Fig D). The multiple fragments detected in the wild species indicate that Dasheng is also abundant in these genomes (Fig 2D).

View larger version (108K): In this window In a new window Download PPT slide   Figure 2. Autoradiograph of Dasheng display with DNAs from O. sativa and other Oryza species. For all reactions, the transposon-specific primer was Pr2. (A) Dasheng display with DNAs from the IR64 x Azucena doubled haploid mapping population using adapter primer MseI + T; (B) Dasheng display with DNAs from Azucena and Nipponbare, two japonica cultivars, using adapter primer BfaI + C; (C) Dasheng display with DNAs from Bs125 and PI1439404, two indica cultivars, using adapter primer MseI + A; (D) Dasheng display with DNAs from eight other Oryza species using adapter primer MseI + TG.

The chromosomal location of polymorphic bands was determined by integrating segregation patterns into a previously constructed framework map consisting of 432 SSRs (TEMNYKH et al. 2001 ). The map was based on a population of doubled haploid lines, derived from a cross between IR64 (indica) and Azucena (japonica). In this study, a total of 215 bands (128 from Azucena and 87 from IR64) from six primer-enzyme combinations were assigned to all 12 chromosomes (Fig 3). Cloning and sequencing of 20 bands provided confirmation that all fragments were amplified from element-containing loci. For this reason, the mapped bands will be referred to as Dasheng markers.

View larger version (125K): In this window In a new window Download PPT slide   Figure 3. Genetic map of rice (O. sativa) with Dasheng and framework markers. Dasheng markers from Azucena and IR64 are in red and green, respectively. Dasheng markers with a ripple of LOD > 2.0 were integrated into the framework map (in boldface type). Dasheng markers that cosegregate with a framework marker with absolute linkage are connected to this framework marker by a horizontal or slanted line. Vertical lines indicate possible intervals for Dasheng markers that are mapped with low LOD scores. Centromeres are indicated by purple boxes. The position of centromeres in this map is based on TEMNYKH et al. 2001 , except for that in chromosome 10, which is based on CHENG et al. 2001A . Also shown for chromosome 11 is a diagram of the distribution of heterochromatic regions (indicated by solid ovals; the open circle in the middle represents the centromere; CHENG et al. 2001B ).

Dasheng markers cluster around all centromeres and on the long arm of chromosome 11. We define a cluster as three or more elements mapping to the same site or to adjacent loci with an average distance of <1 cM. On the basis of this definition, >50% (120) of the elements were clustered in regions that account for only 3% of the total map distance. The largest clusters of markers were on chromosomes 4, 8, 9, and 12, which all correspond to small chromosomes containing prominent blocks of highly condensed chromatin (FUKUI and IIJIMA 1991 ). The correlation of Dasheng clusters and the distribution of heterochromatin is best seen in chromosome 11, where significantly more elements were observed in the distal region on the long arm than in the pericentromeric region (Fig 3). The distal region of chromosome 11 is one of the most heterochromatic regions in the rice genome (CHENG et al. 2001B ).

FISH analysis:

The mapped elements represent only 20% of the Dasheng family. FISH analysis was performed to ascertain whether the entire family shows similar clustering. To this end, an internal fragment of Dasheng (Fig 1) was used as a FISH probe with chromosomes prepared from cv. Nipponbare and Zhongxian 3037, an indica cultivar (Fig 4). In agreement with the mapping results, the majority of the FISH signal concentrated in pericentromeric regions with the most intense signals located on several small chromosomes. One of the small chromosomes with an intense signal was unambiguously identified as chromosome 4 on the basis of its distinctive arm ratio. This chromosome was previously found to contain one of the most heterochromatic regions in the rice genome (CHENG et al. 2001B ). The absence of an exceptionally large cluster of elements on our genetic map could be due to the use of different strains for the genetic mapping vs. the cytogenetic analysis. Alternatively, since the genetic map reflects only polymorphic insertion sites, most of the elements on chromosome 4 may not be polymorphic in this mapping population.

View larger version (34K): In this window In a new window Download PPT slide   Figure 4. FISH analysis of Dasheng distribution in rice mitotic chromosomes. Dasheng probes were detected by fluorescein isothiocyanate-conjugated antibiotin antibody (green); chromosomes were stained with propidium iodide (red). Arrows point to the strong signal of Dasheng on the short arm of chromosome 4 in Nipponbare (see text for details).

Distribution of Dasheng on chromosome 1:

At the time of this study, 30% of rice genomic sequence was publicly available, including almost the entire chromosome 1. To provide a direct physical measure of how densely clustered the elements are on chromosome 1, the positions of all Dasheng were determined from the genomic sequence. The actual distribution of Dasheng elements on a single chromosome permits a determination of whether the apparent clustering of Dasheng in pericentromeric regions on the genetic map might instead be an artifact of the lower recombination rate in these regions (MOORE and SHERMAN 1975 ). In other words, 1 cM may contain far more DNA around the centromeres, and this would give the appearance of clustering on a genetic map even if the insertion frequency is the same, in physical terms, as that in the gene-rich chromosome arms. To test this notion, we dissected chromosome 1 into pericentromeric regions and chromosome arms and calculated both physical:genetic distance and insertion frequency of Dasheng elements in different regions (see MATERIALS AND METHODS for details). Consistent with the low recombination ratio in pericentromeric regions, the ratio of physical:genetic distance was roughly three times higher (660 kb DNA/cM) in pericentromeric regions compared to chromosome arms (206 kb DNA/cM; P < 0.05). However, more significant is the variation in the insertion frequency, which was about five times higher in the pericentromeric regions than in the arms (1.9 vs. 0.4 elements/Mb DNA, P < 0.01). These data confirm the higher density of Dasheng elements in pericentromeric regions.

Dasheng elements are not nested:
LTR retrotransposons are commonly found in large clusters in the genomes of grasses. In many instances, these clusters are composed of LTR retrotransposons inserted into other members of the same family (like BARE-1; SHIRASU et al. 2000 ) or into elements of other families (SANMIGUEL et al. 1998 ). Two rice LTR elements, RIRE3 and RIRE8, were previously found to be nested (KUMEKAWA et al. 1999 ). Although the density of Dasheng in the rice genome (1000 copies/430 Mb; this study) is comparable to that of BARE-1 in barley (14,000 copies/4800 Mb; VICIENT et al. 1999 ), nested insertions of Dasheng elements were not observed. Only 6 out of 109 Dasheng elements are located within 10 kb of another Dasheng element, and the shortest distance between two Dasheng elements was 1.6 kb. As such, the clustering of Dasheng is unlikely to be due to a self-insertion preference, as has been observed for some retrotransposons and MITEs (HIGASHIYAMA et al. 1997 ; JIANG and WESSLER 2001 ).

Since the pericentromeric regions are enriched in repetitive sequences, including transposable elements (DONG et al. 1998 ; LANGDON et al. 2000 ; NONOMURA and KURATA 2001 ), the clustering of Dasheng could also be attributed to an insertion preference for other repetitive DNA, such as microsatellites or other transposable elements (CHRISTENSEN et al. 2000 ). To address this question, sequences flanking all Dasheng elements in the database were used as queries in computer-assisted searches. Of the 109 elements, 19 were found within an identifiable transposable element, 24 were located within 100 bp of an element, and about one-half were associated with low-copy-number sequences. Among this latter group, none showed significant similarity with a comprehensive database of rice TEs (N. JIANG and S. WESSLER, unpublished data).

In addition, unlike RCS1, RIRE3, and RIRE8, three other high-copy-number LTR retrotransposons in rice (DONG et al. 1998 ; KUMEKAWA et al. 1999 ; LANGDON et al. 2000 ; NONOMURA and KURATA 2001 ), Dasheng elements were not flanked by the RCS2 centromere repeat (DONG et al. 1998 ), indicating that Dasheng is not a centromeric component. However, over half of the 215 Dasheng markers described in this study are located in pericentromeric regions. Since Dasheng elements do not specifically insert into other repetitive sequences, these markers may prove useful in the construction of fine structure maps of rice pericentromeric regions and isolation of genes buried in heterochromatic regions. Other cloning strategies frequently miss such genes.

Recent amplification of Dasheng: Evidence from LTR similarity:
Since the LTR of a single retrotransposon is identical upon insertion (LEWIN 1997 ), sequence divergence between LTRs provides a measure of the time of insertion when an estimate of the nucleotide substitution rate is available (SANMIGUEL et al. 1998 ; BOWEN and MCDONALD 2001 ). The average substitution rate in the adh1 and adh2 loci of grasses (6.5 x 10^-9 substitutions per synonymous site per year) has been used to estimate the time of insertion of maize retrotransposons (GAUT et al. 1996 ; SANMIGUEL et al. 1998 ). In this study, a search of the 100 Mb of publicly available rice sequence led to the identification of 109 Dasheng elements of which 60 were full length (56%), 32 were truncated (28%), and 17 were solo LTRs (16%). Among the 60 full-length elements, 35 (58%) have >99.5% LTR similarity, with 15 being identical. In the discussions that follow, these elements are referred to as "recent." LTR sequence similarity of the other 25 elements varies from 92.7 to 99.1%. By using the same base substitution rate as SANMIGUEL et al. 1998 , we estimate that the Dasheng elements with >99.5% LTR identity (58% of the available full-length elements) inserted within the last 500,000 years. This is a conservative estimate because LTRs evolve more rapidly than coding regions like adh1 and adh2 (SANMIGUEL et al. 1998 ) and because reverse transcription is known to be an error-prone process. Based on a comparison with other high-copy-number LTR elements in rice, the Dasheng family has the highest ratio of elements with identical LTRs (15 out of 109; N. JIANG and S. WESSLER, unpublished data). Thus, Dasheng may have amplified more recently than all other high-copy-number elements in the rice genome.

A hypervariable region and tandem repeats:

A phylogenetic tree was constructed on the basis of the LTR sequences of Dasheng elements and some RIRE2 elements (Fig 5) and used to evaluate whether other structural features of Dasheng correlated with recently amplified elements. Of particular interest were a hypervariable region, the tandem repeats, and the solo LTRs.

View larger version (39K): In this window In a new window Download PPT slide   Figure 5. Phylogenetic analysis of LTR sequences of Dasheng and some RIRE2 elements using the neighbor-joining algorithm from distance matrices. Branch length is proportional to genetic distance. Bootstrap values >50 are indicated as a percentage of 1000 replicates. "Dasheng or RIRE2 solo LTRs" indicates that the sequence similarity of these solo LTRs to the Dasheng consensus is not significantly different from that of RIRE2 consensus by t-test.

As mentioned above, the hypervariable region is located between the PBS and the first tract of tandem repeats (Fig 1). This region consists of a common sequence shared by many or a few elements (no sequence is shared by all elements) and a unique sequence. A similarly organized region of sequence heterogeneity was reported for the Stonor elements of maize (MARILLONNET and WESSLER 1998 ). Interestingly, more than half of the recent elements (19 out of 35) have the same sequence in this region (see Fig 5 for branch lengths of elements labeled with an asterisk), suggesting that the recent amplification of Dasheng could be due largely to the transposition of elements in this subgroup. In addition, the average length of the tandem repeat region in this group is significantly longer than that of other elements (3.7 vs. 2.2 kb, t < 0.001). These data do not permit a determination of whether younger elements have more repeats or if repeats are gradually deleting from the older elements.

Evolution of Dasheng elements: Targeted insertion vs. negative selection:
Having both complete sequences of the element and precise chromosomal locations permits a preliminary determination of whether the clustering of Dasheng elements in the pericentromeric region is due to targeted insertion into the gene-rich arms or postinsertion selection. If Dasheng shows no target site preference, but elements are lost over time from the arms, the arms should contain more recent insertions than the pericentromeric regions. However, no significant difference is seen in the number of recent insertions in arms vs. pericentromeric regions. In chromosomal arms, 6 out of 27 (22%) full-length elements have identical LTRs, whereas in pericentromeric regions, 9 out of 33 (27%) have identical LTRs (² test; P > 0.50).

Origin of solo LTRs:

Solo LTRs are believed to arise from intraelement recombination between transiently paired LTRs (PARKET et al. 1995 ). Recently, the formation of solo LTRs in barley has been proposed as a mechanism that can reverse genome expansion (SHIRASU et al. 2000 ). With 16% of the sequenced elements present as solo LTRs, the Dasheng family provides a unique opportunity to address questions about the formation of solo LTRs by analyzing both their age and their distribution relative to full-length family members.

At least two models can account for the formation of solo LTRs. If it is a stochastic process, older insertions are more likely to have undergone recombination and the distribution of solo LTRs should be no different than the distribution of full-length elements. On the other hand, since solo LTRs arise from intraelement recombination, the frequency of solo LTR formation might correlate with regional recombination frequencies. In this case, there would be relatively fewer solo LTRs in the centromeric and pericentromeric regions where recombination rates are much lower than those in the gene-rich chromosomal arms (MOORE and SHERMAN 1975 ). As can be seen in Fig 5, solo LTRs are associated more often with the longer branches, indicating that they are older, on average, than the full-length elements (0.0437 vs. 0.0187, t < 0.001). In addition, the ratio of solo LTRs to full-length elements in the arms (8:27) is only slightly higher than that in pericentromeric regions (6: 33), and the difference is not significant (P > 0.10 by ² test). Taken together, these data suggest that solo LTR formation in the Dasheng family is probably a stochastic process.

Concluding remarks:
In this study, we characterized an unusual LTR element in rice. As a special category of LTR elements, Dasheng is distinguished by its (1) lack of coding capacity, (2) presence of long tracts of tandem repeats, (3) clustering in heterochromatic regions, (4) high copy number, and (5) recent amplification.

Since Dasheng is among the rice elements of highest copy number and most recent amplification, it is of great interest to know if members of the Dasheng family are still capable of retrotransposition. To date, activity has not been demonstrated for any of the high-copy-number LTR retrotransposons in rice. The only active rice elements (such as Tos17) are present in less than five copies and are activated to retrotranspose by tissue culture (HIROCHIKA et al. 1996 ; AGRAWAL et al. 2001 ; YAMAZAKI et al. 2001 ). In fact, although LTR retrotransposons are the major component of most plant genomes, the only high-copy-number LTR retrotransposon with demonstrated activity is BARE-1 from barley (SUONIEMI et al. 1996 ; JAASKELAINEN et al. 1999 ). As such, it is the only genomic component known to be capable of contributing significantly to genome size variation between populations and related species in plants (KALENDAR et al. 2000 ). Like BARE-1 in barley, Dasheng is a major component of the genome of cultivated rice, O. sativa. In addition, a preliminary survey indicates that Dasheng is probably abundant in all species of the genus Oryza (Fig 2). For these reasons it will be important for future studies to determine whether Dasheng elements are still capable of transposition.

The origin of the Dasheng is also of interest since it is a nonautonomous class 1 element. Nonautonomous transposable elements are widespread in eukaryotic organisms. For DNA elements and non-LTR retrotransposons, the copy number of nonautonomous elements is usually much higher than that of the corresponding autonomous element (KAPITONOV and JURKA 1999 ; FESCHOTTE and MOUCHES 2000 ; INTERNATIONAL HUMAN GENOME SEQUENCING CONSORTIUM 2001). Unlike other classes of nonautonomous elements, only a few high-copy-number LTR elements have been characterized. The only other plant element is the maize Zeon-1, which has a copy number of 6000–32,000 (MEYERS et al. 2001 ). However, unlike Dasheng, Zeon-1 is one of the oldest elements in the maize genome (HU et al. 1995 ; SANMIGUEL et al. 1998 ). As such, it will be difficult, if not impossible, to deduce what autonomous element could be responsible for the amplification of Zeon-1. In contrast, the availability of most of the rice genome sequence coupled with the recent amplification of Dasheng facilitates a comprehensive analysis of autonomous elements that could have given rise to Dasheng. At this time, the RIRE2 family with its related LTRs, as well as the presence of some recently amplified members, is the best candidate. Further studies are underway to establish additional connections between these two LTR element families.

	ACKNOWLEDGMENTS

We thank I. King Jordan (National Institutes of Health and National Library of Medicine, NCBI, Bethesda, MD) and Nathan J. Bowen (National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD) for assistance in sequence and phylogenetic analysis and Xiaoyu Zhang for critical reading of the manuscript. This study was supported by grants from the U.S. Department of Energy (DEFG02-94ER20135) and the National Science Foundation (DBI-0077709) to S.R.W.

Manuscript received December 5, 2001; Accepted for publication April 23, 2002.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

AGRAWAL, G. K., M. YAMAZAKI, M. KOBAYASHI, R. HIROCHIKA, and A. MIYAO et al., 2001  Screening of the rice viviparous mutants generated by endogenous retrotransposon Tos17 insertion. Tagging of a zeaxanthin epoxidase gene and a novel OsTATC gene. Plant Physiol. 125:1248-1257.[Abstract/Free Full Text]

BOWEN, N. J. and J. F. MCDONALD, 2001  Drosophila euchromatic LTR retrotransposons are much younger than the host species in which they reside. Genome Res. 11:1527-1540.[Abstract/Free Full Text]

BUREAU, T. E., P. C. RONALD, and S. R. WESSLER, 1996  A computer-based systematic survey reveals the predominance of small inverted-repeat elements in wildtype rice genes. Proc. Natl. Acad. Sci. USA 93:8524-8529.[Abstract/Free Full Text]

CASA, A. M., C. BROUWER, A. NAGEL, L. WANG, and Q. ZHANG et al., 2000  The MITE family heartbreaker (Hbr): molecular markers in maize. Proc. Natl. Acad. Sci. USA 97:10083-10089.[Abstract/Free Full Text]

CHEN, M. P., P. SANMIGUEL, A. C. DE OLIVEIRA, S. S. WOO, and H. ZHANG et al., 1997  Microcolinearity in sh2-homologous regions of the maize, rice and sorghum genomes. Proc. Natl. Acad. Sci. USA 94:3431-3455.[Abstract/Free Full Text]

CHENG, Z., G. G. PRESTING, C. R. BUELL, R. A. WING, and J. JIANG, 2001a  High-resolution pachytene chromosome mapping of bacterial artificial chromosomes anchored by genetic markers reveals the centromere location and the distribution of genetic recombination along chromosome 10 of rice. Genetics 157:1749-1757.[Abstract/Free Full Text]

CHENG, Z., R. BUELL, R. A. WING, M. GU, and J. JIANG, 2001b  Towards a cytological characterization of the rice genome. Genome Res. 11:2133-2141.[Abstract/Free Full Text]

CHRISTENSEN, S., G. PONT-KINGDON, and D. CARROLL, 2000  Target specificity of the endonuclease from the Xenopus laevis non-long terminal repeat retrotransposon, Tx1L.. Mol. Cell. Biol. 20:1219-1226.[Abstract/Free Full Text]

DONG, F., J. T. MILLER, S. A. JACKSON, G. L. WANG, and P. C. RONALD et al., 1998  Rice (Oryza sativa) centromeric regions consist of complex DNA. Proc. Natl. Acad. Sci. USA 95:8135-8140.[Abstract/Free Full Text]

DUBCOVSKY, J., W. RAMAKRISHNA, P. J. SANMIGUEL, C. S. BUSSO, and L. YAN et al., 2001  Comparative sequence analysis of colinear barley and rice bacterial artificial chromosomes. Plant Physiol. 125:1342-1353.[Abstract/Free Full Text]

FESCHOTTE, C. and C. MOUCHÈS, 2000  Evidence that a family of miniature inverted-repeat transposable elements (MITEs) from the Arabidopsis thaliana genome has arisen from a pogo-like DNA transposon. Mol. Biol. Evol. 17:730-737.[Abstract/Free Full Text]

FUKUI, K. and K. IIJIMA, 1991  Somatic chromosome map of rice by imaging methods. Theor. Appl. Genet. 81:589-596.

GAUT, B. S., B. R. MORTON, B. C. MCCAIG, and M. T. CLEGG, 1996  Substitution rate comparisons between grasses and palms: synonymous rate differences at the nuclear gene Adh parallel rate differences at the plastid gene rbcL. Proc. Natl. Acad. Sci. USA 93:10274-10279.[Abstract/Free Full Text]

GE, S., T. SANG, B.-R. LU, and D.-Y. HONG, 1999  Phylogeny of rice genomes with emphasis on origins of allotetroaploid species. Proc. Natl. Acad. Sci. USA 96:14400-14405.[Abstract/Free Full Text]

GUIDERDONI, E., E. GALINATO, J. LUISTRA, and G. VERGARRO, 1992  Anther culture of tropical japonica x indica hybrids of rice (Oryza sativa L.). Euphytica 62:219-224.

HAN, C. G., M. J. FRANK, H. OHTSUBO, and E. OHTSUBO, 2000  New transposable elements identified as insertions in rice transposon Tnr1.. Genes Genet. Syst. 75:69-77.[Medline]

HARUSHIMA, Y., M. JANO, A. SHOMURA, M. SATO, and T. SHIMANO et al., 1998  A high-density rice genetic linkage map with 2275 markers using a single F₂ population. Genetics 148:479-494.[Abstract/Free Full Text]

HIGASHIYAMA, T., Y. NOUTOSHI, M. FUJIE, and T. YAMADA, 1997  Zepp, a LINE-like retrotransposon accumulated in the Chlorella telemetric region. EMBO J. 16:3715-3723.[Medline]

HIROCHIKA, H., K. SUGIMOTO, Y. OTSUKI, and M. KANDA, 1996  Retrotransposons of rice involved in mutations induced by tissue culture. Proc. Natl. Acad. Sci. USA 93:7783-7788.[Abstract/Free Full Text]

HU, W. M., O. P. DAS, and J. MESSING, 1995  Zeon-1, a member of a new maize retrotransposon family. Mol. Gen. Genet. 248:471-480.[Medline]

HUANG, H. and G. KOCHERT, 1994  Comparative RFLP mapping of an allotetraploid wild rice species (Oryza latifolia) and cultivated rice (O. sativa). Plant Mol. Biol. 25:633-648.[Medline]

Initial sequencing and analysis of the human genome. (2001) Nature 409:860-921.[Medline]

JAASKELAINEN, M., A. H. MYKKANEN, T. ARNA, C. M. VICIENT, and A. SUONIEMI et al., 1999  Retrotransposon BARE-1: expression of encoded proteins and formation of virus-like particles in barley cells. Plant J. 20:413-422.[Medline]

JIANG, J., B. S. GILL, G. L. WANG, P. C. RONALD, and D. C. WARD, 1995  Metaphase and interphase fluorescence in situ hybridization mapping of the rice genome with bacterial artificial chromosomes. Proc. Natl. Acad. Sci. USA 92:4487-4491.[Abstract/Free Full Text]

JIANG, N. and S. R. WESSLER, 2001  Insertion preference of maize and rice miniature inverted repeat transposable elements as revealed by the analysis of nested elements. Plant Cell 13:2553-2564.[Abstract/Free Full Text]

KALENDAR, R., J. TANSKANEN, S. IMMONEN, E. NEVO, and A. H. SCHULMAN, 2000  Genome evolution of wild barley (Hordeum spontaneum) by BARE-1 retrotransposon dynamics in response to sharp microclimatic divergence. Proc. Natl. Acad. Sci. USA 97:6603-6607.[Abstract/Free Full Text]

KAPITONOV, V. V. and J. JURKA, 1999  Molecular paleontology of transposable elements from Arabidopsis thaliana. Genetica 107:27-37.[Medline]

KUMAR, A. and J. L. BENNETZEN, 1999  Plant retrotransposons. Annu. Rev. Genet. 33:479-532.[Medline]

KUMAR, S., K. TAMURA, I. B. JAKOBSEN and M. NEI, 2001 MEGA2: Molecular Evolutionary Genetics Analysis Software, Arizona State University, Tempe, AZ.

KUMEKAWA, N., H. OHTSUBO, T. HORIUCHI, and E. OHTSUBO, 1999  Identification and characterization of novel retrotransposons of the gypsy type in rice. Mol. Gen. Genet. 260:593-602.[Medline]

LANDER, E. S., P. GREEN, J. ABRAHAMSON, A. BARLOW, and M. J. DALY et al., 1987  MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1:174-181.[Medline]

LANGDON, T., C. SEAGO, M. MENDE, M. LEGGETT, and H. THOMAS et al., 2000  Retrotransposon evolution in diverse plant genomes. Genetics 156:313-325.[Abstract/Free Full Text]

LEWIN, B., 1997 Genes VI. Oxford University Press, New York.

LI, Z. Y., S. Y. CHEN, X. W. ZHENG, and L. H. ZHU, 2000  Identification and chromosomal localization of a transcriptionally active retrotransposon of Ty3-gypsy type in rice. Genome 43:404-408.[Medline]

MAO, L., T. C. WOOD, Y. YU, M. A. BUDIMAN, and J. TOMKINS et al., 2000  Rice transposable elements: a survey of 73,000 sequence-tagged-connectors. Genome Res. 10:982-990.[Abstract/Free Full Text]

MARILLONNET, S. and S. R. WESSLER, 1998  Extreme structural heterogeneity among the members of a maize retrotransposon family. Genetics 150:1245-1256.[Abstract/Free Full Text]

MCCOUCH, S. R., G. KOCHERT, Z. H. YU, G. S. KHUSH, and W. R. COFFMAN et al., 1988  Molecular mapping of rice chromosomes. Theor. Appl. Genet. 76:815-829.

MEYERS, B. C., S. V. TINGEY, and M. MORGANTE, 2001  Abundance, distribution, and transcriptional activity of repetitive elements in the maize genome. Genome Res. 11:1660-1676.[Abstract/Free Full Text]

MILLER, J. T., F. DONG, S. A. JACKSON, J. SONG, and J. JIANG, 1998  Retrotransposon-related DNA sequences in the centromeres of grass chromosomes. Genetics 150:1615-1623.[Abstract/Free Full Text]

MOORE, C. W. and F. SHERMAN, 1975  Role of DNA sequences in genetic recombination in the iso-1-cytochrome c gene of yeast. I. Discrepancies between physical distances and genetic distances determined by five mapping procedures. Genetics 79:397-418.[Abstract/Free Full Text]

MOORE, G., T. FOOTE, T. HELENTJARIS, K. DEVOS, and N. KURATA et al., 1995  Was there a single ancestral cereal chromosome? Trends Genet. 11:81-82.[Medline]

MYERS, T., 2001  Rice genome consortium will finish ahead of schedule. Nature 409:752.[Medline]

NONOMURA, K. and N. KURATA, 2001  The centromere composition of multiple repetitive sequences on rice chromosome 5. Chromosoma 110:284-291.[Medline]

OHTSUBO, H., N. KUMEKAWA, and E. OHTSUBO, 1999  RIRE2, a novel gypsy-type retrotransposon from rice. Genes Genet. Syst. 74:83-91.[Medline]

PARKET, A., O. INBAR, and M. KUPIEC, 1995  Recombination of Ty elements in yeast can be induced by a double-strand break. Genetics 140:67-77.[Abstract]

SANMIGUEL, P. and J. L. BENNETZEN, 1998  Evidence that a recent increase in maize genome size was caused by the massive amplification of intergene retrotransposons. Ann. Bot. 81:37-44.

SANMIGUEL, P., B. S. GAUT, A. TIKHONOV, Y. NAKAJIMA, and J. L. BENNETZEN, 1998  The paleontology of intergene retrotransposons of maize. Nat. Genet. 20:43-45.[Medline]

SHARMA, S. D., S. R. DHU and P. K. AGARWAL, 2000 Species of genus Oryza and their interrelationships, pp. 311–346 in Rice Breeding and Genetics, edited by J. S. NANDA. Science Publisher, Enfield, NH.

SHIRASU, K., A. H. SCHULMAN, T. LAHAYE, and P. SCHULZE-LEFERT, 2000  A contiguous 66 kb barley DNA sequence provides evidence for reversible genome expansion. Genome Res. 10:908-915.[Abstract/Free Full Text]

SUONIEMI, A., A. NARVANTO, and A. H. SCHULMAN, 1996  The BARE-1 retrotransposon is transcribed in barley from an LTR promoter active in transient assays. Plant Mol. Biol. 31:295-306.[Medline]

SUONIEMI, A., J. TANSKANEN, and A. H. SCHULMAN, 1998  Gypsy-like retrotransposons are widespread in the plant kingdom. Plant J. 13:699-705.[Medline]

TARCHINI, R., P. BIDDLE, R. WINELAND, S. TINGEY, and A. RAFALSKI, 2000  The complete sequence of 340 kb of DNA around the rice Adh1-adh2 region reveals interrupted linearity with maize chromosome 4. Plant Cell 12:381-391.[Abstract/Free Full Text]

TEMNYKH, S., G. DECLERCK, A. LUKASHOVA, L. LIPOVICH, and S. CARTINHOUR et al., 2001  Computational and experimental analysis of microsatellites in rice (Oryza sativa L.): frequency, length variation, transposon associations, and genetic marker potential. Genome Res. 11:1441-1452.[Abstract/Free Full Text]

THOMAS, C. A., 1971  The genetic organization of chromosomes. Annu. Rev. Genet. 5:237-256.[Medline]

TURCOTTE, K., S. SRINIVASAN, and T. BUREAU, 2001  Survey of transposable elements from rice genomic sequences. Plant J. 25:169-179.[Medline]

UOZO, S., H. IKEHASHI, N. OHMIDO, H. OHTSUBO, and E. OHTSUBO et al., 1997  Repetitive sequences: cause for variation in genome size and chromosome morphology in the genus Oryza. Plant Mol. Biol. 35:791-799.[Medline]

VAN DEN BROECK, D., T. MAES, M. SAUER, J. ZETHOF, and P. DE KEUKELEIRE et al., 1998  Transposon display identifies individual transposable elements in high copy number lines. Plant J. 13:121-129.[Medline]

VICIENT, C. M., A. SUONIEMI, K. ANAMTHAWAT-JONSSON, J. TANSKANEN, and A. BEHARAV et al., 1999  Retrotransposon BARE-1 and its role in genome evolution in the genus Hordeum. Plant Cell 11:1769-1784.[Abstract/Free Full Text]

VOS, P., R. HOGERS, M. BLEEKER, M. REIJANS, and T. VAN DE LEE et al., 1995  AFLP, a new technique for DNA fingerprinting. Nucleic Acids Res. 23:4407-4414.[Abstract/Free Full Text]

VOYTAS, D. F., M. P. CUMMINGS, A. KONIECZNY, F. M. AUSUBEL, and S. R. RODERMEL, 1992  copia-like retrotransposons are ubiquitous among plants. Proc. Natl. Acad. Sci. USA 89:7124-7128.[Abstract/Free Full Text]

WAUGH, R., K. MCLEAN, A. J. FLAVELL, S. R. PEARCE, and A. KUMAR et al., 1997  Genetic distribution of Bare-1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP). Mol. Gen. Genet. 253:687-694.[Medline]

XIONG, Y. and T. H. EICKBUSH, 1990  Origin and evolution of retroelements based upon their reverse transcriptase sequences. EMBO J. 9:3353-3362.[Medline]

YAMAZAKI, M., H. TSUGAWA, A. MIYAO, M. YANO, and J. WU et al., 2001  The rice retrotransposon Tos17 prefers low-copy-number sequences as integration targets. Mol. Gen. Genet. 265:336-344.

This article has been cited by other articles:

				K. Kashkush and V. Khasdan Large-Scale Survey of Cytosine Methylation of Retrotransposons and the Impact of Readout Transcription From Long Terminal Repeats on Expression of Adjacent Rice Genes Genetics, December 1, 2007; 177(4): 1975 - 1985. [Abstract] [Full Text] [PDF]

				B. Piegu, R. Guyot, N. Picault, A. Roulin, A. Saniyal, H. Kim, K. Collura, D. S. Brar, S. Jackson, R. A. Wing, et al. Doubling genome size without polyploidization: Dynamics of retrotransposition-driven genomic expansions in Oryza australiensis, a wild relative of rice Genome Res., October 1, 2006; 16(10): 1262 - 1269. [Abstract] [Full Text] [PDF]

Y. Wang, X. Tang, Z. Cheng, L. Mueller, J. Giovannoni, and S. D. Tanksley Euchromatin and Pericentromeric Heterochromatin: Comparative Composition in the Tomato Genome Genetics, April 1, 2006; 172(4): 2529 - 2540. [Abstract] [Full Text] [PDF]