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First published online December 23, 2005; 10.1104/pp.105.066308 Plant Physiology 140:336-348 (2006) © 2006 American Society of Plant Biologists Patterns of Sequence Loss and Cytosine Methylation within a Population of Newly Resynthesized Brassica napus Allopolyploids1,[W]Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (L.N.L., L.O.); and Department of Agronomy, University of Wisconsin, Madison, Wisconsin 53706 (J.C.P., E.L., R.V., T.O.)
Allopolyploid formation requires the adaptation of two nuclear genomes within a single cytoplasm, which may involve programmed genetic and epigenetic changes during the initial generations following genome fusion. To study the dynamics of genome change, we synthesized 49 isogenic Brassica napus allopolyploids and surveyed them with 76 restriction fragment length polymorphism (RFLP) probes and 30 simple sequence repeat (SSR) primer pairs. Here, we report on the types and distribution of genetic and epigenetic changes within the S1 genotypes. We found that insertion/deletion (indel) events were rare, but not random. Of the 57,710 (54,383 RFLP and 3,327 SSR) parental fragments expected among the amphidiploids, we observed 56,676 or 99.9%. Three loci derived from Brassica rapa had indels, and one indel occurred repeatedly across 29% (14/49) of the lines. Loss of one parental fragment was due to the 400-bp reduction of a guanine-adenine dinucleotide repeat-rich sequence. In contrast to the 4% (3/76) RFLP probes that detected indels, 48% (35/73) detected changes in the CpG methylation status between parental genomes and the S1 lines. Some loci were far more likely than others to undergo epigenetic change, but the number of methylation changes within each synthetic polyploid was remarkably similar to others. Clear de novo methylation occurred at a much higher frequency than de novo demethylation within allopolyploid sequences derived from B. rapa. Our results suggest that there is little genetic change in the S0 generation of resynthesized B. napus polyploids. In contrast, DNA methylation was altered extensively in a pattern that indicates tight regulation of epigenetic changes.
A very large number of species are of allopolyploid origin and allopolyploidization is thus one of the most widespread modes of speciation in higher plants. In a successful allopolyploid, two parental genomes must function within a common cytoplasm and be transmitted through meiotic divisions. Developmentally, this process requires both heritable stabilization of the two disparate genomes and the establishment of proper gene transcription levels and timing. One means of this stabilization may be through epigenetic changes that alter chromatin structure and affect gene expression. For example, hypermethylated genes are generally transcribed at a lower level than hypomethylated genes, and the methylation status and gene expression profiles of many allopolyploids differ from their parental genomes at both repetitive and low-copy-number sequences (e.g. Song et al., 1995  ; Chen and Pikaard, 1997a; Kashkush et al., 2002; Madlung et al., 2002, 2005). Genetic changes, including deletions of sequences that are deleterious within the allopolyploid, may also contribute to genome stability (e.g. Feldman et al., 1997). In wheat (Triticum aestivum), DNA sequences that are genome and chromosome specific within established allopolyploids are deleted at high frequency from parental genomes within synthetic allopolyploids (Feldman et al., 1997; Liu et al., 1998b). An analysis of Aegilops sharonensis x Aegilops umbellata F1 plants showed that as much as 14% of the DNA sequences from one parent were eliminated in the synthetic (Shaked et al., 2001). Similar results have been seen in synthesized allopolyploid triticale (Triticum-Secale hexaploids and octaploids) that show that 97% of the Triticum coding sequences, but only 61.6% of the Secale sequences, are maintained after 15 to 35 generations (Ma et al., 2002, 2004).
There has been some debate as to whether observed epigenetic and genetic changes are stochastic or nonrandom. Many of the same sequences that are absent from polyploid bread wheat are eliminated rapidly among a small number of synthetic polyploids generated from different parents (Liu et al., 1998b In this work, we investigate the frequency of genetic and epigenetic changes among 49 newly synthesized Brassica napus allopolyploid lines across a large number of loci. These lines were derived by hybridizing double haploids of Brassica oleracea and Brassica rapa. Thus, they represent independent polyploidization events that are expected to be genetically identical. We investigated DNA sequence and methylation changes among the bulked progeny of S0 polyploids to assay changes in the first generation of polyploidy. Our results show that genetic changes in S0 polyploid genomes are rare. However, epigenetic changes in CpG methylation occur at a much higher frequency, and the frequency and genomic location of methylation changes are tightly regulated.
Loss of Parental Loci within Allopolyploid Genomes
We assayed for genetic changes within 49 unique synthetic polyploids by comparing the EcoRI, HindIII, and DraI restriction fragments of the pooled progeny of B. napus S0 plants with the restriction fragments of their diploid progenitors, B. oleracea (TO1000C) and B. rapa (IMB218A). The 49 S0 lines were generated by interspecific hybridization followed by either colchicine treatment or spontaneous doubling (Fig. 1
). Seventy-six probes were hybridized to each genomic DNA digest. We detected a total of 1,115 fragments within both the B. oleracea and B. rapa genomes. Only 255 (23%) restriction fragments were shared between the two species. Eight-hundred-sixty fragments (77%) were unique to a single species. B. oleracea had a larger number of fragments (718) relative to B. rapa (652). In addition, EcoRI restriction sites were more highly conserved between species than HindIII and DraI restriction sites. Thus, across both species, EcoRI fragments (255) made up a significantly lower number of the unique fragments than HindIII (307) and DraI (298) fragments (P < 0.02). We observed an average of 5.09 fragments per probe-enzyme combination in the B. napus genome. This level of redundancy is consistent with the hypothesis that the two parental genomes are ancient polyploids (Lagercrantz, 1998
Because both parental lines were derived from double-haploid plants, we expected that all fragments detected within the genomes of TO1000C and IMB218A would also be detected within the progeny representing the 49 S0 allopolyploids. Except for a few cases, this expectation was fulfilled. Out of the 54,383 expected parental fragments (54,635 expected minus 252 unreadable fragments), we observed 54,349 (99.94%), within the polyploids (Table I). Figure 2A shows a typical additive pattern of fragments within the diploid and polyploid lines. Figure 2, B to D, illustrates the rare situations (0.06%) where parental genome-specific restriction fragments were not inherited as expected. Among the 34 cases in which a parental fragment was lost (Table I), all 34 were lost from the B. rapa genome (0.18% of all B. rapa-specific fragments).
All but three of the 34 parental fragment changes across the population of nascent polyploids are due to insertions or deletions (indels), and these were not evenly distributed across loci. Two or more restriction digests that detected missing parental fragments indicated an indel. Sometimes, digestion with the third restriction enzyme revealed all parental fragments. In this case, we observed the quantitative loss of the hybridization intensity of one fragment and the increase in the hybridization intensity of another parental fragment (i.e. pX124, HindIII fragments; data not shown). The same change occurred independently 14 times at a locus detected by pX124 (Table II, multilocus genotypes [MGs] 2 and 4). Other changes were rare. A large deletion occurred one time within line EL600B (MG 6) within a sequence similar to pW220 (Table II; Fig. 2B). A small deletion occurred one time in a sequence similar to pW241 in EL9100 (MG 5). Three fragment patterns could not be explained by indels. Probe pW241 detected a novel HindIII fragment in EL3400A and EL2700A (Table II, MG 3 and MG 4; Fig. 2D), but hybridization of DNA restricted by all other enzymes showed parental types. In addition, EL9100A had a pX124 DraI fragment loss that was not accompanied by the loss of any other parental fragments (Table II, MG 5). These fragments may have been generated by star activity or base pair substitution within the recognition sequence. Because of genome redundancy and a lack of knowledge of the restriction sites overlapped by a single probe at a single locus, we cannot tally the proportion of loci that changed among the polyploids. A very conservative estimate is that each probe detected a single locus in each parental genome. Thus, the maximal frequency of indels per locus is 0.2% (16 changes/[49 lines x 2 loci x 76 probes]). Two-thirds of all probes were significantly similar to predicted genes within the Arabidopsis (Arabidopsis thaliana) genome, including the probes pW241, pW220, and pX124.
To confirm and characterize one unique deletion, we cloned and sequenced a 3.6-kb fragment within line EL9100A (Table II, MG 5) that had a 600-bp deletion relative to its 4.2-kb B. rapa parent as identified by pW241 (Figs. 2C and 3A ). Genomic DNA restricted with HindIII of approximately 3,600 bp and 4,200 bp was isolated from both EL9100A and a pooled sample of wild-type polyploid DNA, respectively. We ligated adapters onto the fragments and amplified the allele using primers specific to pW241 and to the adapters. Fragments generated from the primers at the ends of pW241 and the external adapters were the same length in both samples (Fig. 3, B and C), indicating that the deletion occurred within the sequence homologous to pW241. As expected, the internal pW241 fragment derived from EL9100A was 600 bp shorter than the parental control fragment (Fig. 3D). Nucleotide sequences of both fragments were identical at the 5' end except for a guanine at position 491 present within the control and absent from EL9100. From the 5' end starting at position 873, the sequence contained solely guanine-adenine dinucleotides (GpA; Fig. 3E). From the 3' end, both large and small fragments were 99.5% identical for 381 bp. From 1,819 bp toward the 5' end, both sequences were composed of the thymine-cytosine dinucleotide. This result demonstrates that the deletion in EL9100A occurred within a GpA/thymine-cytosine dinucleotide repeat-rich region.
Overall, restriction fragment length polymorphism (RFLP) analyses detected very few genetic differences between parental genomes and synthetic genomes. Over 99% of expected parental fragments were inherited in an additive manner across all 49 allopolyploids (Table I), and 32 of 49 allopolyploids exhibited the expected, parental genotypes (Table II). Sixteen of the remaining 17 allopolyploid progeny each revealed a clear, single indel event. To confirm that these results were representative even if a different screening measure was applied, we also assayed the 49 synthetic genotypes and the parental lines with 30 simple sequence repeat (SSR) primer pairs using DNA from both the S0 plants and S1 pools. Among the S0 plants, the SSR primers amplified 73 different fragments (mean 2.4 and range 1–12 fragments per primer pair that were detected both within a parental genome and within a mix of parental DNA). Of the 3,577 fragments expected among all polyploids (73 codominant fragments x 49 polyploids), 250 were unreadable, but all others were the parental type. Three fragments detected by SSR primer pair snra94 in B. rapa were absent from the control mix of parental genomic DNA due to dominance of the B. oleracea allele. Analyses of the bulked progeny of each S0 plant also revealed no genetic changes. These results confirm both that the genetic structure of B. napus is highly conserved within the S0 and S1 generations and that microsatellite length variation is not a general response to hybridization or polyploidization.
We assayed epigenetic changes within the S0 polyploids by analyzing changes in restriction fragments generated by two isoschizomers, HpaII and MspI. B. oleracea and B. rapa were highly polymorphic for HpaII and MspI recognition sites and differed for 596 fragments. One-hundred-fourteen MspI fragments were present in both genomes, and 103 HpaII fragments were shared as well. Likely because HpaII is sensitive to internal cytosine methylation, we detected a smaller number of polymorphic HpaII fragments than MspI fragments within both B. oleracea (157 versus 172) and B. rapa (129 versus 138). A total of 73 probes were used in this analysis. The three probes that revealed indels (pW241, pW220, and pX124) were not included in the MspI/HpaII survey.
Allopolyploid lines were expected to have the same HpaII and MspI fragment pattern as both diploid parents, indicating no change in methylation status. To test this hypothesis, we made 14,060 pairwise comparisons between polymorphic parental MspI fragments and allopolyploid fragments and 13,524 pairwise comparisons between polymorphic parental HpaII fragments and fragments within the allopolyploids (Table I). Two lines, EL7900A and EL8400C, were missing parent-specific MspI fragments (Table I). Both MspI changes involved the loss of a single MspI fragment derived from B. oleracea and the gain of a novel fragment within a pX144-similar genomic segment. This sequence also changed in our previous analysis of synthetic Brassicas (EC3E12 [Song et al., 1995
Hybridization of DNAs restricted with HpaII showed that cytosine CpG methylation changes between polyploid and diploid genomes were much more frequent than CpCpG changes, and fragments often did not have expected hybridization patterns. In total, 544 of 13,524 parent-specific HpaII fragments present within either B. oleracea or B. rapa were not detected within the polyploids (Table I). A loss of a fragment due to methylation or demethylation may be expected to co-occur with the appearance of a novel fragment. However, within 94% (511/544) of the cases, a loss of a species-specific parental fragment within the allopolyploids was not accompanied by the gain of a novel fragment (Table I). Novel fragments were rare, likely because of genome redundancy (Lukens et al., 2004
Six percent (33/544) of the specific parental HpaII fragment losses were accompanied by the gain of a novel fragment (Table I; Fig. 4C). With this class, we can unambiguously infer whether genes tend to be hyper- or hypomethylated within the polyploid as compared to the diploid. A higher level of CpG methylation within the polyploid genome at a site that was unmethylated within the diploid progenitors would yield novel fragments larger than the missing parental fragments. Sixteen of 17 novel fragments within the B. rapa genome of the allopolyploids were larger than the fragments that were lost, a pattern that differs significantly from the expectation of equality (P < 0.0001). In contrast, within the 14 novel fragments identified in B. oleracea comparisons, eight fragments were larger than the fragments lost, while six novel fragments were smaller. This finding is not significantly different from the expectation of equality (P < 0.42). Thus, de novo CpG methylation primarily occurs within B. rapa homoeologs; while both de novo methylation and demethylation occur within B. oleracea homoeologs.
The distribution of CpG methylation changes across loci shows that specific loci repeatedly underwent epigenetic modification in the S0. Although each of the 73 RFLP probes detected a mean of 7.8 differences in HpaII restriction fragments between the polyploid and diploid genomes, 38 probes (52%) did not reveal any changes across the 49 polyploid lines, and 61 probes (84%) detected fewer changes than the mean (Fig. 5A
). In contrast, pX146 detected 89 changes within the polyploid lines. Eight additional markers, EZ3, pW216, pW219, pW134, pW149, pW170, pX126, and pX128, detected more than 35 changes each. Unexpectedly, noncoding sequences were not over-represented among the nine markers that each accounted for more than 35 changes. The regions of the Arabidopsis genome similar to Brassica probe sequences are given in Supplemental Table I. Brassica probe sequences had only weak similarity to DNA repeats (Supplemental Table II). The variability of genomic changes across probes was much greater than expected (Fig. 5A;
The frequency of epigenetic changes was remarkably uniform across lines (Fig. 5B), consistent with the finding that a small number of loci are preferentially, but not invariably, targeted for epigenetic modification. Methylation changes affected each of the 49 polyploid lines, and each line had eight to 17 fragment changes relative to the diploid progenitors, corresponding to 1.4% to 2.9% of total changes. The mean number of changes per line was 11.61, with a variance of 4.56. The variability of genomic changes across polyploids was much lower than expected (  2 = 18.91, P < 0.0001).
Based on the analysis of lines derived from S0 plants, we suggest that interspecific hybridization and allopolyploidization are associated with rare, targeted indels and more frequent, targeted epigenetic changes. However, indels and epigenetic changes could occur at a similar rate within a population of diploids, could be caused by colchicine treatment, or could be caused by ovule culture. In addition, a B. napus population that had not undergone recent resynthesis may have similar levels of change. To address these questions, we included three controls. First, to assay the frequency of genetic and epigenetic changes between diploids and within the ovule culture, two separate diploid parents were surveyed at all loci. One plant underwent ovule culture; the other was generated from seed. Two TO1000C diploids were identical at all loci. Two IMB218A diploids differed for three novel fragments (at loci detected by pW188 [Hpa II], pW221 [Hpa II], and pW237 [Msp I]). Second, to estimate colchicine effects, we compared two, resynthesized allopolyploid genotypes, EL5700P and EL7800P, that occurred spontaneously (i.e. without colchicine treatment) with resynthesized genotypes EL5700 and EL7800, that were generated by colchicine treatment. Between EL5700 and EL5700P, 1,924 fragments were identical and no fragments differed. Between EL7800 and EL7800P, 1,911 fragments were identical and six fragments differed. Both the diploid pairs and colchicine-treated plants and noncolchicine-treated plants from the same genotype were significantly more similar than were different allopolyploids to each other (
Allopolyploidy is ubiquitous among plants (Levin, 1983 ) and, in many cases, involves large-scale DNA deletions and alterations in cytosine methylation patterns that may cause gene silencing as well as developmental changes (for review, see Wendel, 2000; Liu and Wendel, 2002, 2003; Osborn et al., 2003; Adams and Wendel, 2005). These changes may be highly targeted responses to hybridization and polyploidization or changes may be the result of a less precisely regulated or repeatable process. Studies to date of a small number of individuals have found evidence for both directed and variable changes. In this study, we analyzed a large number of de novo polyploids to investigate the frequency and distribution of the genomic effects that accompany the fusion and doubling of two genomes.
Genetic changes were rare and targeted in Brassica S0 allopolyploids. Of the 76 RFLP probes, 73 detected parental fragments, and the frequency of indels was at most 0.2% per locus. Sixteen indels explained all but three of the 80 fragment differences between the S1 progeny of 49 synthetic S0 polyploids and parental genomes (Table I). One probe (pX124) revealed 14 indels across independently derived individuals, indicating that sequences homologous to this probe are targeted for change (Table II). The estimated frequency of genetic change in S0 Brassica allopolyploids is higher than some species but lower than the frequency of DNA indels from allopolyploids generated within and between the genera Aegilops and Triticum (Ozkan et al., 2001
Several mechanisms could cause the elimination of parental sequences within polyploid genomes, including gene conversion-like events (Wendel et al., 1995
The survey of the population of 49 resynthesized B. napus plants for CpG and CpCpG methylation changes using the 73 RFLP probes that did not show indels revealed 622 total fragment differences between parental and polyploid genomes (Table I). CpCpG methylation changes were very rare, while CpG methylation changes were relatively frequent. In only two cases out of 14,060 pairwise comparisons (0.01%) was a parent-specific MspI fragment lost within a polyploid genome, and only four novel fragments appeared within the polyploids. The maintenance of CpNpG methylation in Arabidopsis involves chromomethylase3 (CMT3; Bartee et al., 2001
Models that take into account the redundancy of the Brassica genome can explain many of the HpaII fragment changes observed within the polyploids. In most cases, the loss of parental fragments did not accompany the gain of a nonparental novel fragment. Out of 544 parent-specific HpaII fragments absent from S0 progeny, 511 (94%) did not co-occur with the gain of a novel fragment. Instead, lost fragments frequently generated an additional fragment that was the same size as an existing fragment. In 38 cases, a nonparental fragment appeared and did not co-occur with the loss of a parental fragment (Table I). Here, it is likely that the polyploid genome had homoeologous loci that generated fragments of the same length. At least one locus was maintained in the parental methylation state after modification of another locus, creating a novel fragment without the loss of a parental fragment. This interpretation is consistent with the highly replicated genome of the Brassicas (e.g. Lukens et al., 2004
In 6% (33/544) of the cases in which a parent-specific fragment was absent from the polyploid, the disappearance of parental fragments coincided with the appearance of a novel fragment (Fig. 4C). This pattern arises because a CpG site that was fully methylated within the diploid progenitor became demethylated or because a site that was demethylated within the diploid progenitor became fully methylated. Within this class, B. rapa loci were preferentially methylated in the polyploid, whereas B. oleracea loci were both methylated and demethylated. De novo cytosine methylation requires both de novo DNA methyltransferases and RNA-silencing genes (Chan et al., 2004
In our large population of resynthesized polyploids, we had the sample size to detect rare epigenetic changes and the distribution of common changes. One finding from this analysis is that CpG methylation changes occurred at a high frequency among some loci (nine probes detected >35 changes) and did not occur among others (38 probes detected no changes). Using smaller populations, several authors also noted the repeatability of epigenetic changes among allopolyploids. For example, Chen and Pikaard (1997b)
Our finding that CpG methylation changes are relatively rare (between 2% and 7%) is consistent with previous analyses of polyploid gene expression and the known targets of methylation. Within maize (Zea mays) polyploids, the expression of most genes maintains a similar per genome level within the polyploid as in the diploid (Guo et al., 1996
Several molecular genetic analyses of polyploids have investigated the genomic changes that occur in the initial and subsequent generations following the formation of a small number of allopolyploids. A large number of differences between the parental and polyploid genomes in wheat are detectable within the interspecific F1 hybrid (Shaked et al., 2001
Both the genetic and epigenetic changes reported here very likely occurred within the S0 generation, perhaps prior to polyploidization. In this analysis, we examined changes within the pooled S1 progeny of self-pollinated, individual S0 plants. Because RFLP and SSR fragments are codominant, the fact that a parental fragment was absent from the bulk of S1 plant DNA indicates that the modification had occurred either within the S0 genome or is a programmed response that occurs many times within or after meiosis of the S0 plant. The most parsimonious explanation is that each modification occurred within an S0 plant prior to flowering. One finding further suggests that most epigenetic changes observed within the S0 progeny had occurred in the S0 hybrid state prior to genome doubling. In our analysis, progeny of two S0 hybrid plants that had one sector treated with colchicine and one sector that spontaneously doubled had either a few or no differences. Prior analyses have shown that hybridization may influence genome epigenetic status (Xiong et al., 1999
By investigating a large number of polyploids, we show that the fusion of distinct genomes in a common nucleus is characterized by targeting specific loci for both genetic and epigenetic change and by maintaining the methylation status of other loci. Interestingly, this targeting is not exact. Many loci are altered infrequently and those loci that are commonly altered are not invariably so. We have previously reported both shared and variable molecular and phenotypic characters in smaller populations of synthesized polyploid Brassicas in later generations (S1–S4 [Song et al., 1995
Plant Material Resynthesized Brassica napus allopolyploid plants (CCAA) were developed by hybridizing plants of Brassica oleracea (CC) genotype TO1000 DH 3, herein TO1000C, and plants of Brassica rapa (AA), genotype IMB 218 DH 1, herein IMB218A. The two parental genotypes (TO1000C and IMB218A) were double-haploid lines of the inbred, self-compatible, and rapid flowering lines of B. oleracea (TO1000) and B. rapa (IMB218). Double-haploid plants were developed by the selection of single plants from microspore culture and the doubling of their chromosomes. Both double-haploid plants were self pollinated, and six male S1 B. rapa plants of IMB218A and 10 female S1 B. oleracea plants of TO1000C were used as parents for interspecific crosses. Siliques were harvested 6 to 7 d after pollination and sterilized siliques were incubated in darkness on White's basal media supplemented with casein at 21°C for 14 d. Embryos were then removed from the siliques, transferred to Murashige and Skoog media, and incubated at 21°C in 16-h-light/8-h-dark cycles. After approximately 21 d, plantlets were transplanted to sterile soil. Forty-nine unique hybrid (CA) plants were generated in this fashion. Embryos from two parental diploid controls were cultured and handled in the same fashion. Separate diploid controls were generated from seed. A total of 51 allopolyploid plants were generated from the 49 hybrid plants using colchicine (Fig. 1). Two of the 49 plants appeared to be mosaics of allopolyploid (CCAA) and hybrid (CA) tissues prior to colchicine treatment. Cuttings were taken from the two regions of both plants and rooted. The hybrid cuttings were subsequently treated with colchicine, generating the allopolyploid plants EL5700 and EL7800. The allopolyploid cuttings generated the plants EL5700P and EL7800P. The two spontaneously doubled lines (EL 5700P and EL 7800P) were excluded from the overall analysis, but used in a later comparison. Of the remaining 47 hybrid plants, eight spontaneously doubled and 39 were treated with colchicine to produce allopolyploid plants. Plants were treated by soaking roots in 0.3% colchicine for 2 h.
To compare rates of cytosine methylation between a control group of polyploids and de novo polyploids, we determined cytosine methylation differences within two segregating populations of double-haploid lines. The Udall population was a cross between a natural B. napus (P1804) and a resynthesized B. napus (JU504). The Quijada population was a cross between two natural B. napus lines (P1804 and RV128). The development of these populations for genetic mapping was described previously (Udall et al., 2005
Genomic DNA was extracted from leaf tissue of young plants using the cetyl-trimethyl-ammonium bromide method (Kidwell and Osborn, 1992 Genome walking was utilized to isolate both upstream and downstream genomic DNA fragments homologous to probe sequences (Universal GenomeWalker kit; BD Biosciences). Amplification products were sequenced using fluorescence-based cycle sequencing and electrophoresis separation (Big Dye Sequencing kit and ABI Prism 377; ABI).
Thirty A or C genome- and chromosome-specific SSR primer pairs (developed by D. Lydiate and A. Sharpe, personal communication) were used to assay genome structure within the synthetic allopolyploids. Fragments were amplified in 10-µL reactions with 1x buffer, 0.2 mM deoxyribonucleotide triphosphate, 0.3 µM of both primers, 2.0 mM MgCl2, 0.5 units Taq polymerase, 40 ng genomic DNA, and 0.1 µL of [
Differences in restriction fragments and SSR amplification products between parental, diploid genomes, and the DNA of S1 allopolyploid plants were tallied to estimate the number of genetic and epigenetic changes between the parental and S0 genomes. The absence of a parental fragment in the allopolyploid was counted as a fragment loss. The presence of a fragment within allopolyploids that was absent from both diploid parents was counted as a gain (as in Table I). Hybridizations of HindIII, DraI, and EcoRI restriction digests were used to identify indels (see "Results"). To estimate the number of epigenetic changes, we compared parental and allopolyploid HpaII and MspI fragments among loci with no evidence of genetic changes. Differences between the S1 allopolyploids and parental fragments for MspI fragments are indicative of changes in CpCpG cytosine methylation. Differences between HpaII fragments at loci that do have parental MspI fragment types are indicative of changes in CpG cytosine methylation.
All analyses were done with the statistical software R (R Development Core Team, 2004 Sequences from RFLP probes have been deposited in GenBank (accession nos. DT469117–DT467171 for the pX probes and CZ 906364–CZ906485 for the pW probes). Sequences were compared with the RefSeq GenBank records NC_003070.4 (ch 1), NC_003071.3 (ch2), NC_003074.4 (ch3), NC_003075.3 (ch4), and NC_003076.4 (ch5) using BLASTn with default parameters. Sequences were also compared with Arabidopsis and Brassica transposable elements downloaded from The Institute for Genomic Research Brassicaceae Repeat Database. All bioinformatics analyses were done with Bioperl.
We thank Dr. Nicole Riddle and two anonymous reviewers for their comments on this manuscript. Received May 28, 2005; returned for revision September 21, 2005; accepted November 1, 2005.
1 This work was supported by the National Science Foundation Plant Genome Program (grant no. 0077774 to T.C.O.), and by the Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Agriculture and Food (grants to L.L.). 
2 These authors contributed equally to the paper.
3 Present address: Division of Biological Sciences, Life Sciences Center, University of Missouri, Columbia, MO 65211.
4 Present address: Seminis Vegetable Seeds, Inc., 37437 State Highway 16, Woodland, CA 95695. The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) are: Lewis N. Lukens (llukens{at}uoguelph.ca) and J. Chris Pires (piresjc{at}missouri.edu).
[W] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.066308. * Corresponding author; e-mail llukens{at}uoguelph.ca; fax 519–763–8933.
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= 0.05). Only 48 of 1,176 (0.04) possible pairwise comparisons between polyploid lines had fewer than six fragment differences and only 1 of 1,176 (<0.001) possible pairwise comparisons had no differences. Finally, to test the possibility that cytosine methylation may vary naturally within the polyploid B. napus, we analyzed DNA of 50 individuals from two mapping populations, one derived from natural x natural and the other derived from natural x resynthesized B. napus parents (Udall et al., 2005
where n is the number of counts, s2 is the variance estimate, and m is the estimate of the mean, will follow a chi-squared distribution with n – 1 degrees of freedom. 
