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American Society of Plant Biologists Sequence Elimination and Cytosine Methylation Are Rapid and Reproducible Responses of the Genome to Wide Hybridization and Allopolyploidy in WheatDepartment of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100, Israel 3 To whom correspondence should be addressed. E-mail avi.levy{at}weizmann.ac.il; fax 972-8-9344181
Interspecific or intergeneric hybridization, followed by chromosome doubling, can lead to the formation of new allopolyploid species. Recent studies indicate that allopolyploid formation is associated with genetic and epigenetic changes, although little is known about the type of changes that occur, how rapidly they occur, and the type of sequences involved. To address these matters, we have surveyed F1 hybrids between diploid species from the wheat (Aegilops and Triticum) group and their derived allotetraploids by screening a large number of loci using amplified fragment length polymorphism and DNA gel blot analysis and by assaying the extent of cytosine methylation. We found that sequence elimination is one of the major and immediate responses of the wheat genome to wide hybridization or allopolyploidy, that it affects a large fraction of the genome, and that it is reproducible. In one cross between Ae. sharonensis x Ae. umbellulata, 14% of the loci from Ae. sharonensis were eliminated compared with only 0.5% from Ae. umbellulata, with most changes occurring in the F1 hybrid. In contrast, crosses between Ae. longissima x T. urartu showed that sequence elimination was more frequent after chromosome doubling. Alterations in cytosine methylation occurred in  13% of the loci, either in the F1 hybrid or in the allopolyploid. For eight of nine bands that were isolated, the sequences that underwent elimination corresponded to low-copy DNA, whereas alterations in methylation patterns affected both repetitive DNA sequences, such as retrotransposons, and low-copy DNA in approximately equal proportions.
The duplication of genomes (polyploidy), either of the same genome (autopolyploidy) or of diverged genomes (allopolyploidy or amphiploidy), is a major force of evolution. It is thought that most living eukaryotes have undergone one or more polyploidization events during their history (Spring, 1997  ; Wolfe and Shields, 1997). One interpretation of the success of polyploidy is that it releases one set of alleles from selective pressure, thus facilitating the acquisition of new functions and buffering the mutation load. In the case of allopolyploidy, the fixation of permanent heterozygosity (Stebbins, 1971; Allard et al., 1993) has the potential to offer a substantial heterozygote advantage. Despite these potential benefits, polyploidy is an enormous challenge with regard to the orchestration of gene expression, DNA replication, and chromosome pairing. How the new polyploid genome deals with such genomic stress is a significant, albeit poorly understood, question.
In plants, it is estimated that polyploidy occurred in the history of
Previous works showing allopolyploidy-induced genetic and epigenetic alterations have focused on selected sequences such as rDNA genes (Sasakuma et al., 1995
Frequency of Allopolyploidy-Associated Genetic Alterations as Estimated by AFLP To assess the frequency of allopolyploidy-associated genetic changes in a quantitative and unbiased manner, two different crosses were studied: Ae. sharonensis (SlSl) x Ae. umbellulata (UU) (cross A) and Ae. longissima (SlSl) x T. urartu (AA) (cross B). The F1 hybrids and the allopolyploids derived from each cross were analyzed by AFLP. The banding pattern of the hybrids is expected to yield a pattern that is additive of both parents because parents derive from inbred lines (confirmed in this study) and because AFLP markers are dominant. All of the cases of deviation from such additivity scored in this study were reproducible (see Methods). Deviation was observed for bands present in only one of the parents (polymorphic bands) and absent in the F1 hybrid and/or the allotetraploid. Another, albeit more rare, type of deviation from additivity was the appearance of new bands that were not present in the parents. Using 24 different primer pairs, 1829 bands were obtained in cross A and 1832 bands were obtained in cross B (Table 1). Of these, the number of polymorphic bands was 373 in cross A and 346 in cross B. In cross A, 20 bands were absent in both F1 and the allotetraploid, suggesting that these bands disappeared at the hybrid level (Figure 1) . Similarly, in cross B, 13 bands disappeared in both F1 and allotetraploid plants. In another group of loci, bands that were present in the parents and in the F1 hybrid disappeared in the allotetraploid plants: there were five such bands in cross A and 28 in cross B. Interestingly, band disappearance was not random: all 20 bands that had disappeared in F1 in cross A originated from Ae. sharonensis, and in cross B, of 13 bands that disappeared in F1, 12 were from Ae. longissima. For bands that were eliminated only in the allopolyploid, there was also a preferential elimination of bands from Ae. sharonensis in cross A (four of five bands that disappeared originated from Ae. sharonensis), whereas in cross B, there was no strong bias for elimination (10 bands in Ae. longissima and 18 in T. urartu). Overall, the percentage of bands that disappeared can be estimated only out of the polymorphic bands because of the dominant nature of AFLP. In cross A, 14% of the bands (24 of 171) from Ae. sharonensis disappeared compared with 0.5% (one of 202) from Ae. umbellulata. Most of the bands had disappeared in the F1 hybrid (20 versus five in the allopolyploid). In cross B, 12.2% of the bands (22 of 180) from Ae. longissima disappeared compared with 11.4% (19 of 166) from T. urartu, and the timing of band disappearance was later than in cross A (i.e., more events occurred in the allopolyploid [28 events] than in the F1 hybrid [13 events]).
In addition to band disappearance, which was the most common event observed, we found five cases, among the 1832 AFLP bands analyzed in cross B, of new bands that were not seen in the parents that appeared in F1 and disappeared again in the allopolyploid (Figure 1, band C). Similarly, we found seven cases of bands that were present in the parents, disappeared in F1, and reappeared in the allotetraploid (data not shown). These deviations from additivity were probably caused by changes in methylation of the EcoRI site. Alterations in AFLP patterns caused by methylation changes are described in the MSAP analysis. Here, we have focused on the analysis of bands that disappeared in F1 and/or the allopolyploid.
DNA Gel Blot Validation of the AFLP-Detected Genetic Alterations Nine bands that disappeared, either in F1 or in the allopolyploid (all from cross B), were isolated from acrylamide AFLP gels (Figure 1) and used as probes for DNA gel blot analysis. The results of this analysis are shown in Table 2 and are described below. Eight of the nine bands originated from low copy DNA, and one was from high-copy DNA. Sequence analysis did not reveal any significant similarity for any of the nine sequences. In two cases, that of AFLP-isolated fragments 4 and 5 (AIF4 and AIF5), validation could not be determined, either because there was no polymorphism between the parents (AIF4) or because the band originated from high-copy DNA (AIF5) and discrete bands could not be analyzed. In all seven cases in which validation could be tested, the band elimination observed by AFLP was confirmed by DNA gel blot analysis (Figure 2) . Note that, for all of the probes tested, the pattern of elimination was reproducible in three different S1 plants of each allotetraploid. This finding, as well as the reproducibility of migration patterns in the parents (data not shown), indicates that the deviation from additivity was not caused by the heterozygosity of the parents. Moreover, some of the enzymes used in the DNA gel blot analysis (DraI and EcoRV) are not particularly sensitive to methylation and band disappearance was not associated with the appearance of new bands, suggesting that for the cases studied here, the band disappearance in AFLP was not caused by cytosine methylation. In summary, AFLP is a robust and high throughput means to assess the induction of genomic rearrangements. Elimination was the most frequent genomic rearrangement; it was reproducible and could not be attributed to heterozygosity or methylation.
Frequency of Allopolyploidy-Associated Epigenetic Alterations as Estimated by MSAP The enzyme HpaII is sensitive to methylation of either cytosine residue at the recognition site (5'-CCGG), whereas its isoschizomer MspI is sensitive only to methylation of the external cytosine. Therefore, methylation of the internal cytosine would lead to a differential cleavage by the two isoschizomers and thus to the appearance of different MSAP fragments in the sequencing gel loaded with the amplification products from EcoRI+MspI versus EcoRI+ HpaII digests (Figure 3) . Another, albeit less frequent, cause of the differential banding pattern, namely the presence of fragments in the EcoRI+HpaII digests and their absence in the EcoRI+MspI digests, has been observed (Figure 3) and has been attributed to hemimethylation of the external C, resulting in blocking of MspI digestion (McClelland et al., 1994 ).
Seven pairs of selective primers produced 501 clear bands in the two parental lines Ae. sharonensis (genome SlSl) and T. monococcum ssp aegilopoides (genome AmAm) (Table 3). Each of the bands represented a recognition site cleaved by one or both of the isoschizomers. Of the 501 resolved bands, 159 were methylated in one or both parental lines (Table 3). The two parents showed essentially the same degree of methylation, namely 30.2% for Ae. sharonensis and 33.2% for T. monococcum ssp aegilopoides (data not shown). Therefore, on average, 31.7% (159 of 501) of the 5'-CCGG sites in the parental line genome were cytosine methylated in the leaves (Table 3).
The two parental lines, the F1 hybrid, and the allotetraploid were compared for their methylation patterns (Table 4, Figure 3). As mentioned for the AFLP analysis of genetic alterations, we expected that F1 and the allotetraploid would have the combined methylation pattern of both parents. Any deviation from additivity was considered an alteration in methylation pattern related to the F1 and/or the allotetraploid situation. Five major classes were identified among the methylated fragments (Table 4): (1) 21 bands (13.2% of all 159 methylated sites) were monomorphic, that is, they had the same pattern of methylation among parental lines, the F1 hybrid, and the allotetraploid; (2) 117 bands (73.5% of all methylated sites) showed differential cytosine methylation between the parents but no deviation from additivity in F1 or the allotetraploid; (3) three bands (1.8% of all methylated sites) showed differential cytosine methylation patterns between parents and F1 hybrids, but methylation was restored to the parental type in the allotetraploid; (4) 11 bands (6.9% of all methylated sites) showed alteration in cytosine methylation starting in the F1 hybrid that was maintained in the allotetraploid (interestingly, of those, 10 were from aegilopoides and one was from sharonensis); and (5) seven bands (4.4% of all loci) showed methylation alteration in the allotetraploid but not in the F1 hybrid.
DNA Gel Blot Validation of the MSAP-Detected Epigenetic Alterations DNA gel blot analysis was performed to confirm the methylation patterns detected by MSAP using 12 MSAP-isolated fragments (MIFs) as probes. These included five fragments (of 11 total) that showed methylation alteration in both the F1 hybrid and the allotetraploid and all seven fragments that showed methylation alteration in the allotetraploid only (Table 4). Total DNA from leaves was digested with either HpaII or MspI. All 12 fragments were sequenced and analyzed for similarity to known sequences in the database. Three of the five fragments that showed methylation alteration in the F1 hybrid (MIFs 1 to 5) showed high similarity to repetitive DNA sequences and highly repeated retrotransposons, and two fragments showed no significant similarity to known sequences (Table 5).
Sequence analysis of the seven fragments that showed methylation alteration in the allotetraploid (MIFs 6 to 12) showed no significant similarity to known sequences (Table 5). DNA gel blot analysis of these fragments showed that three of these seven fragments were high-copy number and the rest were low-copy number (Table 5). Methylation alteration in the allotetraploid was validated for all four low-copy fragments using DNA gel blot analysis. An example is shown in Figure 4A for MIF8 as a probe (Table 5). Furthermore, the four low-copy fragments were hybridized to membranes containing the same DNA samples digested with methylation-insensitive restriction enzymes. In these cases, there was no alteration in the DNA gel blot hybridization pattern. An example is shown in Figure 4B using MIF8 as a probe. Similarly, altered methylation in F1 and/or the allopolyploid was validated for all five low-copy probes tested. These results show perfect agreement between DNA gel blot analysis and methylation alteration in the allopolyploid detected by MSAP. The high-copy probes led to a smear on the autoradiogram, leaving us unable to assess methylation alteration (data not shown).
Note that using MSAP, we also recovered fragments that were not differentially methylated but that showed band disappearance in F1 and/or the allopolyploid, as was found using AFLP (Figures 1 and 2). DNA gel blot analysis of two such bands showed elimination in the allopolyploid using both membranes that included DNA samples digested by isoschizomers (Figure 5A) and membranes that included the same DNA samples digested with nonsensitive restriction enzymes (Figure 5B). These two bands were both low copy and showed no significant similarity to known sequences.
In previous works, we have shown that a limited set of loci could undergo rapid elimination in newly synthesized allopolyploids from the wheat group (Feldman et al., 1997 ; Liu et al., 1998b; Ozkan et al., 2001). These findings were based on the analysis of fragments that had the unexpected property of not being present in all genomes of polyploid wheat (chromosome-specific or genome-specific sequences). In this study, we have addressed the frequency of genetic and epigenetic changes in interspecific and intergeneric F1 hybrids and in newly synthesized allopolyploids and have sequenced some of the loci involved. This was done by analyzing a large and unbiased sample of wheat loci using the AFLP method (Vos et al., 1995) or the derived MSAP method (Xiong et al., 1999). The banding patterns obtained by both methods were found to be reproducible; moreover, AFLP or MSAP bands showing unexpected inheritance in the F1 hybrid or the allopolyploid were isolated, sequenced, and used as probes for DNA gel blot analysis. In all cases, the DNA gel blot data corroborated the AFLP or MSAP data, indicating that AFLP and MSAP provide reliable and high throughput methods to test for genetic and epigenetic changes, respectively.
The Observed Genetic and Epigenetic Alterations Are Not the Result of Heterozygosity
Frequency and Timing of Genetic and Epigenetic Alterations
Alterations in cytosine methylation also were associated with the hybrid and allopolyploid condition (Table 4, Figure 3). Few events (1.8%) were found whereby the methylation pattern was altered in F1 but was restored to the parental type in the allopolyploid. Most events of methylation pattern alteration had occurred in the F1 hybrid (6.9%) and were maintained in the allopolyploid or occurred only in the allopolyploid (4.4%). These results are higher than reported for methylation alterations in F1 of intraspecific crosses; in rice, Xiong et al. (1999)
Underlying Mechanism of Rapid and Reproducible Allopolyploidy-Associated Sequence Elimination
At this time, we have no support for or against the other mechanisms, and experiments are under way in our laboratory to determine the deletion or recombination breakpoints and thus gain more insight into the underlying elimination mechanism. In any case, this work shows that sequence elimination is the major and immediate response of the wheat genome to wide hybridization and allopolyploidy, that it can affect a large fraction of the genome (up to 14% of the polymorphic fragments), and that it is reproducible. In this respect, allopolyploidy somehow fits McClintock's definition of a genomic shock that "initiates a highly programmed sequence of events within the cell that serves to cushion the effects of the shock" (McClintock, 1984
Plant Material The plant material for this study consists of three different combinations of newly synthesized allotetraploids, F1 hybrids, and their homozygous diploid parents. The first cross involved Aegilops sharonensis (genome SlSl) x Ae. umbellulata (genome UU), the second cross involved Ae. longissima (genome SlSl) x Triticum urartu (genome AA), and the third cross involved Ae. sharonensis (genome SlSl) x T. monococcum ssp aegilopoides (genome AmAm). The synthetic allotetraploids were obtained after colchicine treatment of the F1 plants. Chromosome number was determined in root tip mitosis of the F1 hybrids and the allotetraploids. The parental lines and the synthetic allopolyploids are maintained in the plant collection of our laboratory, and their production is described by Ozkan et al. (2001) .
Amplified Fragment Length Polymorphism (AFLP) Analysis The adaptor sequences were as follows: MseI adaptors, 5'-TACTCAGGACTCAT-3' and 5'-GACGATGAGTCCTGAG-3'; EcoRI adaptors, 5'-CTCGTAGACTGCGTACC-3' and 5'-AATTGGTACGCAGTC- TAC-3'. Preselective amplification was performed with primers complementary to the core of the adaptor sequences; the EcoRI preselective primer was 5'-GACTGCGTACCAATTCA-3', and the MseI preselective primer was 5'-GATGAGTCCTGAGTAAC-3'. The polymerase chain reaction (PCR) contained 50 ng of restricted-ligated DNA, 50 ng of EcoRI preselective primer, 50 ng of MseI preselective primer, 1 unit of Taq DNA polymerase, 2 µL of 10 x Taq DNA polymerase buffer (1 x Taq DNA polymerase buffer is 50 mM Tris HCl, pH 8.0, 100 mM NaCl, 0.1 mM EDTA, 1 mM DDT, 50% glycerol), 2 µL of 25 mM MgCl2, and 2.5 mM deoxynucleotide triphosphates in a final volume of 20 µL. The PCR conditions were 20 cycles of 30 sec at 94°C, 60 sec at 56°C, and 60 sec at 72°C. After preamplification, the PCR products were diluted 1:20 with 190 µL of double-distilled water.
Selective amplification was performed with the 32P-end-labeled EcoRI selective primers and the unlabeled MseI selective primers. The labeling reaction contained 50 ng of EcoRI selective primer, 100 µCi of The selective amplification reaction contained 50 ng of template DNA from the preselective amplification, 1 ng of labeled EcoRI selective primer, 5 ng of MseI selective primer, 1 unit of Taq DNA polymerase, 2 µL of Taq DNA polymerase buffer, 2 µL of 25 mM MgCl2, and 2.5 mM deoxynucleotide triphosphates in a final volume of 20 µL. The PCR cycles were one cycle of 2 min at 94°C, 30 sec at 65°C, and 2 min at 72°C, followed by 10 cycles each of annealing temperature of 1°C less than the former one, and 25 cycles of 1 sec at 94°C, 30 sec at 56°C, and 2 min at 72°C. The PCR products were mixed with 20 µL of dye (98% formamide, 10 mM EDTA, 0.1% bromphenol blue, and 0.1% xylene cyanol). The denatured PCR products were separated on 6% denaturing polyacrylamide (20:1 acrylamide:bisacrylamide, 7.5 M urea, and 1 x Tris-borate-EDTA buffer, pH 7.8) standard sequencing gel (43 cm in length) at 55 W for 1.5 hr. Gels were dried and exposed to x-ray film (Fuji Photo Film, Tokyo, Japan) for 6 to 12 hr at room temperature.
Methylation-Sensitive Amplification Polymorphism (MSAP) Analysis
Scoring of AFLP and MSAP bands
Cloning of Fragments Subjected to Genetic or Epigenetic Alterations and Validation by DNA Gel Blot Analysis
Sequence Analysis
We thank an anonymous referee for constructive comments. This work was supported by a United States–Israel Binational Science Foundation fund grant. H.O. was supported by the Turkish Council of Higher Education and by a short-term European Molecular Biology Organization fellowship. K.K. and H.S. were supported by a fellowship from the Feinberg Graduate School for Ph.D. and M.Sc., respectively.
1 These two authors have contributed equally to this work. 
2 Current address: Department of Field Crops, Faculty of Agriculture, University of Cukurova, Adana 01330, Turkey. Received March 1, 2001; accepted June 8, 2001.
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Abstract
INTRODUCTION
-32P-ATP, 10 units of T4 polynucleotide kinase, and 10 µL of 5 x polynucleotide kinase buffer (1 x polynucleotide kinase buffer is 50 mM Tris HCl, 1 mM DDT, 0.1 mM EDTA, 50% glycerol [v/v], pH 7.5) in a final volume of 50 µL. Samples were incubated at 37°C for 1 hr and then heated to 70°C for 10 min. The sequence of the selective primers is similar to that of the preselective primers, with the addition of two variable nucleotides at the 3' end. The AFLP primers are identified by the last three specific nucleotides of the selective primers, namely EcoRI+ANN primers and MseI+CNN primers. 
