Global genomic analysis of transposable element distributions of both natural (En/Spm, Ac–Ds, and MuDR/Mu) and modified (RescueMu) types was performed by fluorescence in situ hybridization (FISH) on somatic chromosomes coupled with karyotyping of each chromosome. In lines without an active transposable element, the locations of silent En/Spm, Ac–Ds, and MuDR/Mu elements were visualized, revealing variation in copy number and position among lines but no apparent locational bias. The ability to detect single elements was validated by using previously mapped active Ac elements. Somatic transpositions were documented in plants containing an engineered Mutator element, RescueMu, via use of the karyotyping system. By analyzing the RescueMu lines, we found that transposition of RescueMu in root-tip cells follows the cut-and-paste type of transposition. This work demonstrates the utility of FISH and karyotyping in the study of transposon activity and its consequences.

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TRANSPOSON activity was first noted by Barbara McClintock in her seminal work in maize. She observed variegated plants and found that this pattern of variegation was transmissible (MCCLINTOCK 1950, 1958). She subsequently characterized the Activator–Dissociation (Ac–Ds) system that has served as a model for transposon study to the present. The dissociation (Ds) element was so named because it caused chromosomal breaks when an Ac element was present. Because of her cytological expertise with maize chromosomes, she was able to recognize that the breakage site was mobile. Except for McClintock's pioneering work, cytology has not been extensively applied to the study of transposons.

Since the discovery by McClintock, many transposable elements, including the autonomous Ac (FEDOROFF et al. 1983; POHLMAN et al. 1984), En/Spm (PEREIRA et al. 1986; MASSON et al. 1987), and MuDR (CHOMET et al. 1991; HERSHBERGER et al. 1991; QIN et al. 1991) elements of maize as well as those in many diverse taxa, have been cloned and their role in genome evolution is now appreciated (BENNETZEN 2000, 2005). Because techniques were not available to directly visualize the chromosomal location of transposable elements, subsequent work to characterize their activity relied on genetics and the tools of molecular biology. Using these approaches, it has been possible to learn about many aspects of transposon behavior. However, these approaches are complicated by the presence of inactive elements in the maize genome that are identical, or nearly so, to active autonomous elements and nonautonomous responders. Such inactive elements cannot be easily studied genetically. Because molecular cytological techniques have progressed considerably in recent years, their application could enable additional approaches in transposon studies. In particular, the sensitivity of fluorescence in situ hybridization (FISH) detection has improved, allowing detection of very small genomic targets (KATO et al. 2006; WANG et al. 2006).

In this study, we demonstrate the use of FISH to visualize the physical location of active and silent transposons. We document the genomic position of silent En/Spm, Ac–Ds, and MuDR/Mu transposable elements in three standard maize inbred lines. The utility of FISH to map new Ac insertions is demonstrated. The transgenic Mu element RescuMu (RAIZADA et al. 2001) is detected using FISH, and its transposition behavior in root-tip somatic cells is analyzed.

Plant materials:

Kernels of RescueMu progeny from Grid G parents (RAIZADA et al. 2001) were provided by G. Nan and V. Walbot (Stanford University). Kernels of Ac stocks (KOLKMAN et al. 2005) were provided by L. Conrad, J. Kolkman, and T. Brutnell (Boyce Thompson Institute, Cornell University). Twin sector ears containing P-vv were provided by W. F. Sheridan (University of North Dakota). Standard maize inbred lines, KYS, B73, and Oh43, and the B73 background with B chromosomes were taken from stocks previously used for cytological characterization (KATO et al. 2004; LAMB et al. 2005).

Southern hybridization:

Maize genomic DNA isolation, probe labeling, and Southern hybridization were performed as described (YU et al. 2006b). An 1-kb RescueMu-specific probe was prepared from pBluescript II KS(+) plasmid (Stratagene, La Jolla, CA) by BspHI digestion followed by gel isolation as previously described (RAIZADA et al. 2001).

Chromosome preparation and FISH:

Somatic chromosome spreads were produced as previously described (KATO et al. 2004) except the concentration of cellulase was increased from 2 to 4% in the enzymatic mixture for root-tip digestion and slides were used within 4 hr of preparation. Probe hybridization was carried out at 55° for 12–24 hr and washed in 2x SSC for 20 min (KATO et al. 2004). Chromosomes were stained with 4',6-diamidino-2-phenylindole (DAPI) containing Vectashield mounting media (Vector Laboratories, Burlingame, CA). Signals were captured with an epifluorescence microscope and a cooled coupled device camera. Images were modified using Adobe Photoshop as previously described (KATO et al. 2004; LAMB and BIRCHLER 2006).

FISH probes:

The MuDR probe was prepared from a plasmid (pCLMu9.6) containing MuDR that was provided by V. Walbot (Stanford University). The Ac probe was prepared from DNA produced by PCR using a plasmid (4W-1) containing a full-length Ac element that was provided by J. Kolkman and T. Brutnell (Boyce Thompson Institute, Cornell University) as a template and the following primers were designed from the Ac element: AC18Rev (5'-GTTTTTAATCGGGATGATCCCGTTTCGT) and JGp3'Rev (5'-ACCGATACGATCCGGTCGGGTTAAAGTC). The En/Spm probe was prepared from DNA produced by PCR using a plasmid template (SLJ12522) containing a full-length En/Spm Rev element provided by N. Fedoroff (Pennsylvania State University). Three pairs of primers were used to amplify the En/Spm transposase by PCR and the products were combined stoichiometrically for use as a FISH probe: (1) spm210f (5'-TTCTACAGCCGTCGTGCTTCTTCT) and spm3286r (5'-ATTAGCCGCATAGCGATCTGGGAA); (2) spm2220f (5'-AGACAGCAAAGCAAATGAGGTGGC) and spm5696r (5'-CGGGTAGGCGAATCGCAAACAAAT); and (3) spm7940r (5'-ATGGGAAGCCTCCATAACAGCACA) and spm4279f (5'-TTGCAGCTACAACAAACAGCGGAG). A pBluescript II KS(+) plasmid was used as a probe for the detection of RescueMu. The p1 gene probe was prepared from three plasmids provided by S. Chopra (Pennsylvania State University): pWRG57, which contains 6242 bp of P1-wr 5' upstream sequence; pF2cDNA, which contains the 1670-bp p1 gene cDNA; and pSA204, which contains the 1420-bp insert from intron 2 of the p1 gene. The inserts of pWRG57, pF2cDNA, and pSA204 were released by SalI, BamHI/XhoI, and KpnI/PstI digestions, respectively, and were recovered by gel isolation.

All probes were labeled using a nick translation reaction with a high concentration of polymerase I (40–50 units/microgram DNA) that incorporates nucleotide analogs (TexasRed-dUTP for the transposons and AlexaFluor488-dUTP for p1) that have covalently attached fluorescent molecules (KATO et al. 2006). In addition to several discrete sites of hybridization, the MuDR probe also weakly labeled the nucleolar organizer region (NOR) regions. In our experience, the direct use of plasmids to make FISH probes occasionally results in this pattern of NOR labeling, probably caused by bacterial genomic DNA that contaminates plasmid preparations.

A karyotyping probe cocktail was prepared as described (KATO et al. 2004) and used simultaneously with the RescueMu probe (labeled with TexasRed-dUTP, pseudocolored white in Photoshop). The karyotyping cocktail contains the following probes labeled with the indicated fluorochrome: Cent4 and 5S ribosomal DNA 2-3-3 clone, Cy5-dUTP (pseudocolored red in Photoshop); CentC, fluorescein-dUTP; microsatellite 1-26-2 clone, fluorescein-dUTP; telomere-associated sequence pMTY9ER, AlexaFluor488-dUTP; NOR-173 clone, fluorescein-dUTP; and the 180-bp heterochromatin knob sequence, coumarin-dUTP. A simplified cocktail containing CentC (fluorescein-dUTP), microsatellite 1-26-2 (fluorescein-dUTP), subtelomeric repeat 4-12-1 (fluorescein-dUTP), and the 180-bp knob repeat (coumarin-dUTP) was used to identify chromosomes for the B73, Oh43, and KYS inbred lines. Final probe concentration was 15–30 ng/µl of each probe in the mixture and 5 µl of probe mixture was added to each chromosome preparation.

FISH is a robust method for genome karyotyping (KATO et al. 2004), characterization of chromosomal aberrations (YU et al. 2006a), and evolution (HAN et al. 2005). In this study, two changes were made to the basic FISH method to allow consistent detection of small targets of 3 kb. First, we found that an increase of the cellulase concentration in the root-tip digestion mix from 2 to 4% significantly reduced background fluorescence after hybridization. Second, FISH was performed on samples immediately after chromosome spreading and UV crosslinking because storing chromosome preparations resulted in reduction of signal intensity over time. These improvements allowed us to consistently detect small targets, such as natural and engineered transposable elements (Ac–Ds, En/Spm, MuDR/Mu, and RescueMu).

Visualization of an active Ac element at the p1 locus:

Insertion of an active Ac element into the p1 gene gives rise to the P1-vv allele (BARCLAY and BRINK 1954; LECHELT et al. 1989). Disruption of the p1 gene causes loss of pigmentation of the pericarp (the maternal layer of tissue that surrounds the mature kernel) and the cob. Because excision of the Ac element during ear development restores p1 function and gives rise to sectors of red pigmentation in the pericarp, it is possible to track the P1-vv allele genetically. Additionally, a Ds insertion into the r1 gene (r1-sc:m3) allows Ac transposase activity to be monitored in the endosperm by variegated purple pigmentation (ALLEMAN and KERMICLE 1993). To determine if the active Ac element could be detected by FISH, p1 gene sequences were labeled in green (AlexaFluor488-dUTP) and simultaneously hybridized with a red (TexasRed-dUTP) Ac probe to chromosome spreads from root tips germinated from colorless and purple-variegated seeds of an ear from a P1-vv/p1 tester (P1-wr; r/r) x p1 tester (P1-wr; r1-sc:m3/r1-sc:m3) cross. In kernels lacking functional Ac transposase, two chromosomes had a bright green signal while kernels with purple variegation had one bright green signal and one weaker green signal. Some alleles of the p1 locus are composed of multiple copies of the p1 gene (CHOPRA et al. 1998) and the intensity of FISH hybridization should reflect the number of copies present. The brighter intensity of the P1-wr signal allowed the tester allele to be distinguished from the P1-vv allele. At P1-vv, but not at P1-wr, the red and green signals colocalized (Figure 1).

View larger version (25K): In this window In a new window Download PPT slide   FIGURE 1.— Cytological visualization of an active Ac element at the p1 locus. Chromosome spreads were hybridized with p1 sequences (green) and a near full-length Ac element (red) and counterstained with DAPI (blue). Chromosomes in A–C are from a plant heterozygous for the P1-vv allele and the p1 tester allele P1-wr and homozygous for the r1-sc:m3 reporter. Those in D–F are from a P1-wr homozygote. In A, both p1 alleles are labeled and can be distinguished by signal intensity (green). Ac signal colocalizes with the p1 signal at the P1-vv locus (less intense green signal) but not with the p1 tester allele (p1-wr). The gray values for the p1 signal (B and E) and the Ac signal (C and F) are displayed below the respective merged image. The insets show a magnified view of the P1-vv locus with the arrows indicating the site of p1 hybridization. In the inset in A, the Ac (red) and P1-vv (green) signals are shown together (light yellow).

 

En/Spm, Ac–Ds, and MuDR/Mu in B73, Oh43, and KYS:

Using the En/Spm transposase sequence, near full-length Ac, or full-length MuDR elements as FISH probes, the location of individual transposable elements was visualized on somatic chromosomes for three standard maize inbred lines: B73, KYS, and Oh43 (Figure 2). A small number of additional FISH probes were included in the hybridization mixture as a karyotyping cocktail so that each chromosome could be identified. Additionally, the signal pattern on the maize supernumerary B chromosome is displayed. Within each line, the locations of the transposon FISH signals are constant although FISH signals are occasionally undetectable or vary in intensity from cell to cell. Because the size of transposable elements is near the lower limit of FISH detection, the occasional missing or weak signal could be due to detection failure although element loss through transposition is also a possible explanation. Appearance of hybridization signals at new locations was not observed.

View larger version (61K): In this window In a new window Download PPT slide   FIGURE 2.— En/Spm, Ac–Ds, and MuDR/Mu hybridization pattern of B73, Oh43, and KYS inbred lines. Somatic metaphase chromosome preparations from three lines—B73 (A, D, and G), Oh43 (B, E, and H), and KYS (C, F, and I)—were hybridized with a collection of probes to identify each chromosome (180-bp knob repeat in blue; CentC and a "TAG" microsatellite repeat in green) and the En/Spm transposase (A–C), full-length Ac (D–F), or full-length MuDR element (G–I) probes (red). Chromosomes from a single representative spread of each type were cut and arranged in pairs. The transposon signals are presented in gray values below the merged images. Spreads containing the maize supernumerary chromosome were hybridized with CentC (green) and a transposon probe (red). The B chromosomes are displayed in row J.

 
Although the positions of transposon FISH signals varied among inbred lines, the total copy number was similar for each element. The En/Spm element hybridized to more locations than the other two elements. The B73 and KYS lines had 36 sites of En/Spm hybridization while Oh43 had 43. There were also more sites of Ac–Ds hybridization in B73 and KYS than in Oh43 and 10–12 copies in B73 and KYS compared to only 5–6 copies in Oh43. As a comparison, Southern blot hybridization using a portion of the Ac element as a probe detected 11 clear bands in a W22 inbred line carrying the P1-vv allele (KOLKMAN et al. 2005). About 15–16 copies of MuDR/Mu homologous elements were detectable in all three lines. Southern blot hybridization also reveals Mu homologous sequences in nonmutagenic lines (LISCH et al. 1995).

There was variation for chromosomal position of transposons among the inbred lines examined in this study. For example, on chromosome 5 in Oh43, no Ac signal was detected while chromosome 5 in B73 had strong signals on the short arm and KYS had sites on the short arm and proximal sites on the long arm. Chromosomes 7–10 did not have a bright Ac signal in any of the three lines. All of the probes produced some signals that were intense and others that were weak. Weaker signals are expected for some silent and nonautonomous elements that would have diverged sequences or deletions. The positions of the silent Ac–Ds and MuDR/Mu transposon copies did not show any apparent bias toward particular chromosome regions as signals are located at various positions along the chromosomes. The En/Spm signals were also fairly uniformly distributed although several chromosomes had clusters of signals near the middle region. On the B chromosome, an En/Spm signal was present at or near the centromere and in multiple sites on the B long arm in the proximal euchromatin. Two Ac–Ds signals and one MuDR/Mu signal were detected at distal locations on the B long arm.

In addition to the detectable sites of transposable elements, there are small nonautonomous elements residing in the genome. Many nonautonomous elements that have been recovered consist of little more than the inverted repeats (RUBIN and LEVY 1997). These elements would not be detectable as they are below the current level of sensitivity for the FISH technique.

Detection of mapped active Ac elements:

Once the pattern of FISH signals is determined for a given line, it should be possible to detect alterations in that pattern that arise from novel transposition events. A collection of maize stocks containing an active Ac element at different mapped positions has been described (KOLKMAN et al. 2005). The new Ac insertions in these stocks originated from the P1-vv allele or from "bti97156::Ac," which has an insertion on chromosome 5, bin 5.04 (SINGH et al. 2003). FISH was performed on chromosome spreads from 14 stocks in this collection using the Ac probe in combination with a cocktail of FISH probes that allows the chromosomes to be identified (Figure 3). In each Ac-containing stock, all of which were generated in the W22 inbred line background, when the FISH signal pattern was compared to the typical pattern for the W22 inbred, a new signal was observed in a location corresponding to the previously reported genetic location. The absence of signals at the original Ac location is because all stocks had the original site removed in subsequent genetic crosses (KOLKMAN et al. 2005). Additionally, FISH was used to confirm the location of Ac-im that was recently mapped to 7.02 (CONRAD and BRUTNELL 2005) (Figure 3L).

View larger version (55K): In this window In a new window Download PPT slide   FIGURE 3.— FISH detection of active Ac elements. FISH was performed on somatic chromosomes from 14 stocks of Ac element insertions that were independently generated in the W22 inbred line. The stocks used and the bin in which the Ac insertion is mapped are as follows: (A) P1-vv, 1.03 (1S); (B) mon00200::Ac, 1.10/1.11 (1L); (C) bti00191::Ac, 2.02 (2S); (D) mon00178::Ac, 3.05 (3-centromeric bin); (E) bti00220::Ac, 3.04 (3S); (F) bti03616::Ac, 3.09 (3L); (G) mon00150::Ac, 4.08/4.09 (4L); (H) mon00212::Ac, 5.01 (5S); (I) mon00238::Ac, 5.06 (5L); (J) bti00194::Ac, 5.07/5.08 (5L); (K) bti95076::Ac, 6.05 (6L); (L) Ac-im, 7.02 (7L); (M) mon00092::Ac, 9.07/9.08 (9L); and (N) bti9924::Ac, 10.1/10.2 (10S). All "bti" insertions originated from the original P-vv allele while "mon" insertions originated from "bti97156::Ac," which has an insertion on chromosome 5 (bin 5.04). The Ac probe (red) was used in combination with a cocktail of FISH probes that allowed each chromosome to be identified. The cocktail consists of the 180-bp knob (blue), CentC (green), "TAG" microsatellite (green), the subtelomeric repeat 4-12-1 (green), and Cent4 (pseudocolored white). The chromosome arm containing the new signal is indicated on the left. A typical chromosome from a line without the new insertion is shown in column 1 next to the first case of transposition for each chromosome. Column 4 shows the hybridization pattern of the indicated chromosome lines containing mapped Ac insertions. The Ac hybridization signals from columns 1 and 4 are shown as gray values in column 2 and 3, respectively. In A, the active Ac element located at the original position on chromosome 1 at the p1 locus (P1-vv) can be seen and is compared to a chromosome 1 without the P1-vv insertion.

 

Cytological detection of RescueMu by FISH and its validation by Southern hybridization:

The sensitive FISH technique allowed us to detect three classic maize transposable elements (Ac–Ds, En/Spm, and MuDR/Mu). For each of these transposon families, multiple silent copies exist in the maize genome. Because FISH labels these nonfunctional elements, it is necessary to account for all the typical FISH signals before new transposition events can be confirmed. To more easily track individual elements, an engineered transposon, RescueMu, was studied.

The RescueMu system was created by RAIZADA et al. (2001) by modifying a nonautonomous Mu1 element through insertion of an 3-kb bacterial plasmid, pBluescript. Because the transposase binds to the inverted repeats of the nonautonomous Mu1 element, RescueMu transposes in the same fashion as natural nonautonomous elements. To detect the RescueMu element, the plasmid, pBluescript, was used as a FISH probe. No endogenous sequences with similarity to pBluescript are present in maize so this probe is highly specific for RescueMu.

A comparative analysis between FISH and Southern hybridization was used to validate our FISH results. The copy numbers were determined by Southern blot analysis as well as by FISH, demonstrating that we can accurately detect the RescueMu element with the pBluescript plasmid probe (Figure 4). For example, both heterozygous (Figure 4A, lanes 1, 3, and 5) and homozygous (Figure 4A, lane 4) RescueMu loci were detected (Figure 4B, D, E, and F).

View larger version (90K): In this window In a new window Download PPT slide   FIGURE 4.— Validating RescueMu FISH with Southern hybridization. (A) Plant G33-32 containing two RescueMu loci was selfed and genomic DNA was isolated from five individuals. The DNA was digested with HindIII and Southern blots were hybridized with a 32P-dCTP-labeled 1.0-kb bla gene (ampicillin resistance gene) fragment probe from pBluescript. Ethidium bromide staining of the gel indicated a similar total DNA in each lane (not shown). Two bands among the progeny that correspond to the two RescueMu sites in the parent (arrows a and b) were detected. The five individuals have the following constitution: 1, a/–; 2, b/b; 3, a/–, b/b; 4, a/a; and 5, a/–; indicating heterozygous and homozygous insertions on the basis of band intensity. (B–F) Chromosome spreads from the five individuals were hybridized with the Bluescript probe. Images in B–F correspond to lanes 1–5 on the Southern blot in A. RescueMu insertions are indicated by arrows. Bar, 10 µm.

 

Somatic transposition of RescueMu:

Because there are many copies of transposable elements in the maize genome, it is difficult to track particular elements by molecular methods such as Southern hybridization. Using FISH, loss of an element at a given location can be clearly visualized. However, the ability to detect a new insertion could be simplified by using an in vitro modified and reintroduced element. Thus, to study the transposition process, RescueMu transgenic maize lines were analyzed by FISH to detect somatic transpositions in root tips. Cells from 10 individuals from three RescueMu lines were analyzed (Table 1), and the chromosomal positions in each observed cell were recorded (supplemental Table 1 at http://www.genetics.org/supplemental/). The following observations were made.

View this table: In this window In a new window   TABLE 1 Chromosomal locations of RescueMu in somatic cells of different maize seedling root tips

 
First, we observed the segregation of the RescueMu element in the progeny of G1-1x73, which are representatives from a population used for maize gene discovery and which contain copies of the autonomous MuDR (FERNANDES et al. 2004). Four RescueMu insertions (3S, 5S, 10L, and 10L) were observed. While individuals 2, 3, 4, and 6 inherited all four insertions, three sites (3S, 10L, and 10L) were transmitted to individual 7, two sites (10L and 10L) were observed in individual 1, and only one site (5S) was found in individual 5. These materials are from an outcross of G1-1 by a B73 plant in Grid G (V. WALBOT, personal communication), which was heterozygous for the four sites.

Second, somatic excision was observed as suggested by the failure to observe specific insertions in a fraction of the cells in a root tip. The two 10L sites were used as an example to examine this issue. These two RescueMu sites were present in most of the cells on the long arm of chromosome 10 (10L:10L) of individuals in the progeny of the cross G1-1x73 that inherit this chromosome. However, an average of 38.6% of the cells from the six individual root tips showed the loss of one of the 10L sites, most likely by somatic excision. The calculated somatic excision rate from the six individuals ranges from 19.0% (individual 1) to 57.1% (individual 4) (supplemental Table 1 at http://www.genetics.org/supplemental/). These values are likely to vary due to the size of the excision sector in the sampled root tip. It is not possible to determine which of the two 10L sites is lost without additional markers on the chromosome, but the two sites serve as an internal technical control for each other with regard to the ability to detect the RescueMu sequences in a particular cell.

Third, six new RescueMu insertions were observed in two lines (G1-1x73 and G33-32) (Tables 1 and 2). The appearance of a new insertion in a particular cell was always correlated with the disappearance of a progenitor site of hybridization (Table 2). For example, in root tips of G1-1x73 individual 4, 53 individual cells were analyzed among which a new site on chromosome 9L was found, and in the same cell a loss of signal at a chromosome 10L locus was observed (Figure 5). These results provide evidence that the RescueMu transposition in root tips is consistent with the "cut-and-paste" mode of transposition.

View this table: In this window In a new window   TABLE 2 Transpositions of RescueMu in somatic cells

 

View larger version (34K): In this window In a new window Download PPT slide   FIGURE 5.— An example of RescueMu transposition in somatic cells. Chromosome spreads from two different cells from plant G1-1x73#4 that have different RescueMu insertion sites consistent with a cut-and-paste transposition are shown. Chromosomes are labeled with the pBluescript KS plasmid (white) and a karyotyping probe cocktail to identify the chromosomes. The karyotyping cocktail contains the following probes: Cent4 (red), 5S rRNA (red), CentC (green), microsatellite 1-26-2 (green), pMTY9ER telomere-associated sequence (green), NOR-173 (green), and the 180-bp knob (blue). In A and B, RescueMu signals alone are shown in gray values and the chromosome identification signals are shown in C and D. The merged images are in E and F. The RescueMu signals are indicated by arrows. The numbers in E and F indicate the chromosomes that have RescueMu sites. Bar, 10 µm. RescueMu insertion sites are magnified (x4) in E1–3 and F1–3. In the first cell (A, C, E), four insertions—one on 3S, one on 5S, and two on 10L—were detected. In the second cell (B, D, F), a new RescueMu insertion on 9L was detected. This new site was correlated with the loss of one of the 10L insertions.

 
Fourth, a stable RescueMu element was identified in one line (G9-2). Many individual cells were examined from two individuals containing this insertion and no instances of RescueMu transposition were observed (supplemental Table 1 at http://www.genetics.org/supplemental/). The stability of this RescueMu site was most likely caused either by mutations of the terminal inverted repeat sequence of RescueMu or by the lack of MuDR/Mu transposase, which catalyzes the transposition.

In this work, we demonstrate that DNA transposons can be visualized in cytological preparations. Simultaneous application of a chromosome identification (karyotyping) cocktail of FISH probes with transposon probes provided a genomewide view, allowing the chromosomal location of the silent transposons of detectable size to be determined. When the positions of silent transposable elements are compared among the B73, Oh43, and KYS inbred lines, several differences are observed. Such variation complicates attempts to follow active Ac elements using Southern blot or FISH approaches. However, these differences could serve as cytological markers to track recombination in the progeny of hybrids. Comparing Ac–Ds or MuDR/Mu signals to classic molecular markers could assist in integrating the genetic map and cytological maps for different inbred lines. In addition to documenting copy number differences among maize varieties, the technique could be used to study the phenomenon of activation of silent elements by irradiation or chromosomal breakage (PETERSON 1960; MCCLINTOCK 1965). The method could also be used to test for the mobilization of transposable elements in wide crosses or in newly generated allopolyploids (MADLUNG et al. 2005).

Insertion preference for transcriptionally active regions has been described for Ac–Ds and En/Spm when expressed in heterologous systems (KOLESNIK et al. 2004; KUMAR et al. 2005). Also, in maize, examination of Ac–Ds insertion sites revealed a preference for insertion into unique sequences (COWPERTHWAITE et al. 2002). However, the positions of the silent Ac and En/Spm transposon copies did not show any apparent bias toward known chromosomal regions in the three lines examined with one apparent exception: En/Spm elements were more abundant in the euchromatic portion of the B chromosome. The maize supernumerary B chromosome is dispensable and has virtually no phenotypic effects on plants harboring it (CARLSON 1978). The B chromosome long arm is composed of a heterochromatic block followed by a euchromatic portion and four large heterochromatic distal regions (CARLSON 1978). However, the apparent euchromatic localization on the B chromosome may be coincidental, given that only a few elements were present and a single isolate of the B chromosome was sampled.

After the pattern of hybridization for a given line is determined, transposition of active elements to new sites in the genome can be detected. Collections of maize stocks containing active Ac insertions at different locations could serve as starting material to recover insertional mutants for genes with known locations (AUGER and SHERIDAN 1999; CONRAD and BRUTNELL 2005; KOLKMAN et al. 2005). This approach takes advantage of the tendency of the Ac element to transpose to nearby locations by allowing researchers to choose stocks where the active Ac is close to the gene of interest, thereby reducing the number of subsequent transposition events that would need to be screened for insertion in the desired location (AUGER and SHERIDAN 1999). One drawback to this approach is that it requires mapping large numbers of novel Ac insertions, a time-consuming and tedious process. Because the FISH procedure on somatic chromosomes is simple and fast, it could be used to determine the chromosome arm of new transposition events. This approach would allow the more time-consuming molecular mapping techniques to be limited to only those events that are of interest. The potential of this approach was demonstrated by mapping a new Ac insertion on chromosome 7 (Figure 3L) and a number of RescueMu insertions.

Natural Mutators transpose actively and usually exist at high copy numbers, which complicates their analyses (LISCH et al. 1995). The RescueMu transposon is a modified Mu element containing a pBluescript plasmid sequence that distinguishes it from other MuDR/Mu elements. RescueMu can transpose in both germinal and somatic tissues as do the natural MuDR/Mu elements when the MuDR transposase is supplied (RAIZADA et al. 2001). There are only a few RescueMu copies in the maize genome so it is easy to detect transposition events. Therefore, RescueMu facilitates study of MuDR/Mu behavior. A unique characteristic of Mutator transposons is the replicative transposition during germinal transpositions (LISCH et al. 1995). The replicative transposition mechanism allows the MuDR/Mu elements to accumulate to high copy numbers and is considered to be operative only in germinal cells (RAIZADA et al. 2001). Previous analysis of RescueMu in leaf tissues by directly determining flanking sequences revealed that RescueMu transposition in leaf tissue occurs late in development and suggested a cut-and-paste mechanism for transpositions (RAIZADA et al. 2001). In this study, we used a sensitive FISH method to visualize RescueMu transpositions directly in root-tip somatic cells. The data support a cut-and-paste mechanism for these observations.

In all six cases in which a RescueMu was detected at a new location, exactly one RescueMu had disappeared from an original site. The gain of an extra RescueMu was never observed. The distribution of the six characterized RescueMu transposition cases seems to be random; however, closely linked cases might not be distinguished as discrete sites from the progenitor.

Because this technique permits examination of large numbers of individuals, it could be used to study many aspects of transposon biology. For example, it could be used to determine the genomewide properties of insertion for specific transposon families with regard to preferred sites of integration. In addition, transposons are known to create chromosomal aberrations (ZHANG and PETERSON 2004), which the karyotyping system would allow one to document. Also, the genomewide cytological visualization has the potential to study the activation of quiescent elements, among other uses.

We thank G. Nan and V. Walbot for providing RescueMu materials and the MuDR construct; L. Conrad, J. Kolkman, and T. Brutnell for Ac lines and the Ac construct; W. F. Sheridan for the P1-vv line; N. Fedoroff for the Spm construct; and S. Chopra for p1 constructs. Funding was provided by the National Science Foundation (grant DBI0423898) from the Plant Genome Initiative.

¹ These authors contributed equally to this work.

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