Plant Molecular Biology 43: 387399, 2000. M.A. Matzke and A.J.M. Matzke (Eds.), Plant Gene Silencing.

2000 Kluwer Academic Publishers. Printed in the Netherlands.

387

Genetic and epigenetic interactions in allopolyploid plants

Luca Comai
Department of Botany, Box 355325, University of Washington, Seattle, WA 98195-5325, USA (e-mail: comai@u.washington.edu)

Key words: chromosome evolution, gene silencing, heterochromatin, interspecic hybridization, recombination, transposons

Abstract Allopolyploid plants are hybrids that contain two copies of the genome from each parent. Whereas wild and cultivated allopolyploids are well adapted, man-made allopolyploids are typically unstable, displaying homeotic transformation and lethality as well as chromosomal rearrangements and changes in the number and distribution of repeated DNA sequences within heterochromatin. Large increases in the length of some chromosomes has been documented in allopolyploid hybrids and could be caused by the activation of dormant retrotransposons, as shown to be the case in marsupial hybrids. Synthetic (man-made) allotetraploids of Arabidopsis exhibit rapid changes in gene regulation, including gene silencing. These regulatory abnormalities could derive from ploidy changes and/or incompatible interactions between parental genomes, although comparison of auto- and allopolyploids suggests that intergenomic incompatibilities play the major role. Models to explain intergenomic incompatibilities incorporate both genetic and epigenetic mechanisms. In one model, the activation of heterochromatic transposons (McClintocks genomic shock) may lead to widespread perturbation of gene expression, perhaps by a silencing interaction between activated transposons and euchromatic genes. Qualitatively similar responses, of lesser intensity, may occur in intraspecic hybrids. Therefore, insight into genome function gained from the study of allopolyploidy may be applicable to hybrids of any type and may even elucidate positive interactions, such as those responsible for hybrid vigor. Abbreviations: AFLP-cDNA, amplied fragment length polymorphism of copy DNA; HDGS, homologydependent gene silencing; LTR, long terminal repeat; RFLP, restriction fragment length polymorphism; RT-PCR, reverse transcriptase polymerase chain reaction; WU-BLASTN, Washington University Basic Local Alignment Search Tool, nucleotide comparison algorithm.

Introduction Whereas wild and cultivated allopolyploid plants are well adapted, man-made allopolyploids are typically unstable. Instability refers to the appearance of unexpected changes in phenotype, such as homeotic transformation and lethality, and in genome structure, such as chromosomal rearrangements and changes in the number and distribution of repeated DNA sequences within heterochromatin. These instabilities are presumably eliminated by evolutionary adaptations giving rise to stable species. However, the molecular

mechanisms underlying these adaptive steps remain obscure. This review provides examples of instability and considers the genetic and epigenetic mechanisms that may be responsible for instability. Interspecic hybrids between distant relatives are usually sterile. In 1912, Digby found a fertile plant that appeared spontaneously among the sterile hybrids generated in a cross between two primrose species; this plant had twice the number of chromosomes of its sterile sibs (Digby, 1912). While the signicance of chromosome doubling eluded Digby, Winge, unaware of Digbys results, speculated that speciation could

[ 267 ]

388

Figure 1. Comparison of diploid and allotetraploid hybrids. The parental origin of chromosomes is marked by black and white. A. Production of an allotetraploid hybrid requires a genome duplication step (GD), which can occur in the parents (forming autopolyploids), during meiosis (forming 2N gametes), or after zygote formation (Ramsey and Schemske, 1998). Karyotypic divergence between species prevents normal meiotic pairing of chromosomes at metaphase I in the diploid hybrid, while genome duplication in the allotetraploid hybrid allows normal pairing. Perfect homologous pairing in allotetraploids is often a secondary adaptation (Sears, 1976; Moore, 1998). B. Allopolyploidy hinders recombination between parental genomes. Chiasmata between paired parental chromosomes in a diploid hybrid allow the combination of advantageous parental genome segments while both parental genomes remain xed in the allotetraploid hybrid.

occur by interspecic hybridization followed by chromosome doubling (Winge, 1917). Winge believed that hybrid sterility was caused by unbalanced chromosome sets. He reasoned that upon doubling, a proper pairing partner would be available to each chromosome resulting in fertility (Figure 1A). This prediction was experimentally veried by Clausen and Goodspeed (1925) in tobacco species, and by Karpechenko (1927) in radish and cabbage. It was soon realized that allopolyploids, hybrid species that contain two or more diploid sets of parental genomes, are quite common in nature (Soltis and Soltis, 1993; Leitch and Bennett, 1997; Rieseberg and Noyes, 1998). Allopolyploids represent a special type of hybrid. The two parental genomes in an allopolyploid, called homeologous, undergo little intergenomic recombination and thus maintain their integrity through sexual generations (Figure 1B). Therefore, karyotypic stability is achieved at the expense of the evolutionary exibility provided by unhindered recombination between the parental genomes. In contrast, diploid hybrids freely recombine the two parental genomes, generating progeny with numerous combinations of parental chromosomal segments which can then be xed by selection (Ungerer et al., 1998). The forced maintenance of two integral parental genomes should limit the evolutionary exibility of allopolyploid hybrids. Yet, their widespread distribution implies that allopolyploidy is often advantageous. One factor that favors allopolyploids may be heterosis generated by the combination of homeologous genes (Allard et al.,

1993; Jiang et al., 1998). Another factor may be the instability of new allopolyploids which, although usually deleterious, might generate sufcient phenotypic variation for the exploitation of new ecological niches.

Genomic instability and restructuring The genomes of established allopolyploids can be changed considerably from the parental genomes (Kamm et al., 1995; Wendel et al., 1995; Zhao et al., 1998; Volkov et al., 1999). Since the original parents were not available, conclusions about genomic alteration were based on the structure of parental genomes inferred from the structure of the presumed diploid descendants. Nevertheless, studies with synthetic (man-made) allopolypoids suggest that the observed genomic changes occurred in a burst after allopolyploidization (Song et al., 1995; Feldman et al., 1997; Liu et al., 1998). Two mechanisms that could cause these genomic changes are recombination and transposon activation. Homeologous recombination Homeologous recombination is generally deleterious. Recombination between repeated regions destabilizes the genome by producing deletions and chromosomal rearrangements, while pairing and recombination between homeologous chromosomes hinder the normal progress of meiosis by forming, for example, trivalents

[ 268 ]

389 system has at least two functions: in addition to repairing mismatches produced during replication, it rejects recombination partners when it senses mismatches in recombination-generated heteroduplexes (Figure 3B). As a consequence, failure of mismatch repair increases both the mutation rate (Fishel and Kolodner, 1995; Jiricny, 1998; Prolla, 1998) and homeologous recombination (Bailis and Rothstein, 1990; Selva et al., 1995; Chambers et al., 1996; Hunter et al., 1996), with grave consequences for the function and integrity of the genome. Furthermore, mismatch repair proteins, whose concentration is usually low, can be titrated by an excess of mismatches during incipient recombination between homeologous genomes (Claverys et al., 1983; Humbert et al., 1995). The function of mismatch repair could thus be compromised in interspecic crosses. In a positive feedback loop, this may cause a further increase in homeologous recombination (see below). It is possible that higher expression levels of mismatch repair proteins would be required for stability of allopolyploids than autoploids. Wild-type Salmonella typhimurium is unable to recombine its DNA with that of Escherichia coli, a related bacterium about 80% similar in DNA sequence, even though DNA can be exchanged by conjugation. However, Salmonella mutants decient in mismatch repair were capable of conjugational recombination with E. coli (Rayssiguier et al., 1989). Furthermore, bacteria decient in mismatch repair underwent frequent deletion of DNA anked by homeologous direct repeats (Petit et al., 1991). Collapse of mismatch repair would induce genomic instability in a manner consistent with observations made in allopolyploids (Figure 4). For example, a case of genomic instability in a synthetic Brassica allopolyploid might be explained as a case of homeologous recombination (Song et al., 1995). In this study, three genera with basic diploid genomes (A, B, C) were hybridized in pairwise combinations. In some AB allotetraploids, probes used for restriction fragment length polymorphism (RFLP) mapping hybridized to a new, smallerthan-expected restriction fragment. These new RFLPs were not found in the parental A and B genomes; however, they were identical in size to the RFLP found in the C genome. This peculiar coincidence could be explained by assuming that an ancestral progenitor of the Brassica species had direct repeats in that region of the genome. After speciation of the C genome, a recombination event between the repeats deleted the intervening DNA. Such an event did not occur in the parental B genome, which maintained the two re-

Figure 2. Chromosome pairing in allotetraploids. Normal (left) and aberrant (right) metaphase I gures, corresponding to homologous and homeologous pairing respectively.

and univalents (Figure 2). The occurrence of homeologous recombination depends on the history of allopolyploids. In newly formed allopolyploids, recombination between the homeologous parental genomes can be frequent and may contribute to genome restructuring. On the other hand, homeologous recombination is rare in established allopolyploids. Progressive differentiation of the parental genomes in allopolyploids may prevent homeologous synapsis and recombination, forcing the establishment of a diploid-like meiosis in which the chromosomes of each parental set pair only with their homologues. Additionaly, genetic analysis suggests that certain genes, such as wheat Ph1, can also suppress homeologous pairing (Sears, 1976). The nature of Ph1 is unknown. Mechanisms acting at different levels of chromosome pairing have been postulated (Moore, 1998). Ph1 could encode a local editor that screens pairing partners on a DNA segment by segment basis, allowing recombination only when it recognizes a certain threshold of similarity (Luo et al., 1996). Alternatively, Ph1 could control chromosomal movement or condensation or could recognize chromosomal features other than DNA sequence (Aragon-Alcaide et al., 1997; Mikhailova et al., 1998; Vega and Feldman, 1998a, b). Homeologous recombination in recently formed allopolyploids may play a role in their instability. The destabilizing effect of homeologous recombination might be better understood by considering its relationship to post-replicative DNA mismatch repair. DNA mismatch repair is initiated when a mutS-like protein binds to a mismatched base pair or displaced loop in double-stranded DNA (Modrich and Lahue, 1996; Kolodner and Marsischky, 1999), leading to replacement of a large patch of single-stranded DNA containing the mismatch (Figure 3A). The mismatch repair

[ 269 ]

390

Figure 3. A possible role for DNA mismatch repair in allopolyploidy. A. DNA mismatch repair in eukaryotes is initiated by the binding of a mutS-homologue dimer to the mismatch and proceeds to excision and resynthesis of the mismatched DNA strand. The ladder represents dsDNA. B. A Holliday junction is formed by recombination between two homeologous chromosomes. Because of junction movement (branch migration) complementary homeologous strands form heteroduplexes that contain mismatches (small arrows). Normally, binding of mismatch repair proteins to the mismatches blocks recombination (Rayssiguier et al., 1989; Bailis and Rothstein, 1990; Selva et al., 1995; Chambers et al., 1996). If an excessive number of mismatches are formed, mutS protein could be depleted, allowing homeologous DNA strands to continue the recombination process (Humbert et al., 1995).

peats. Upon AB allopolyploidization, a decrease in mismatch repair function might have allowed the two homeologous repeats to undergo the same recombination event, resulting in a deletion and formation of the C-specic RFLP. Transposon activation Barbara McClintock believed that genomic incompatibilities unmasked by interspecic hybridization are among the causes of genomic shock, a programmed response to stress (McClintock, 1984). She pointed to the difference in repetitive elements between the two parental genomes as a cause of genomic shock, suggesting that hybridization initiated mobilities of these elements. Although this hypothesis has not been directly tested, it is consistent with two phenomena observed in allopolyploids: meiotic drive and chromosome expansion. The terms repetitive elements and transposons are used intercheangeably in this review, and encompass retroelements, DNA transposons, and related heterochromatic repeats. Meiotic drive Meiotic drive refers to the subversion of meiosis by a locus that causes its own preferential segregation. It is common in newly synthesized allopolyploids as well as in diploid hybrids and is associated with heterochroFigure 4. Genomic consequences of recombination between homeologous DNAs. Two examples of genomic changes caused by homeologous recombination. The black and gray arrows represent homeologous sequences. A. Formation of an acentric and a dicentric chromosome. Circles represent centromeres. B. Production of a smaller restriction fragment by deletion of a region anked by homeologous direct repeats (arrows).

[ 270 ]

391 matin and chromosomal breaks. Pollen killer genes, for example, drive their inheritance by causing chromosomal breaks in meiotic products in which they are absent (Endo, 1990). Pollen killer genes are a probable cause of the frequent sterility observed in synthetic allopolyploids. While one or two pollen killer genes can be tolerated, higher numbers are incompatible with survival of the hybrid species bearing them. Although the molecular basis of this phenomenon is unclear, the chromosome-breaking action is reminiscent of that of transposons and may account for at least part of the extensive genome restructuring in allopolyploids. A well studied instance of meiotic drive was discovered in crosses between North American and Latin American maize lines (Rhoades, 1942). In maize, an ancient tetraploid (Whitkus et al., 1992), loci involved in meiotic drive coincide with knobs, polymorphic heterochromatic regions that are detectable cytologically in various chromosomal positions (Buckler et al., 1999). The knobs are activated to drive by the presence of a large heterochromatic region, the knobby arm of abnormal chromosome 10 (Ab10). In the presence of Ab10, each knob assumes neocentromeric activity and is preferentially inherited during megasporogenesis (Yu et al., 1997). Furthermore, because larger knobs result in higher degrees of preferential segregation, the Ab10 meiotic drive system may favor expansion of the heterochromatic repeats that comprise the knobs. The Ab10 interaction with the knob heterochromatin exemplies how the relationship between controlling loci and a family of heterochromatic repeats may contribute to the restructuring of allopolyploid genomes. Expansion of chromosomes The frequent changes in copy number and chromosomal position of repeats (Feldman et al., 1997; Liu et al., 1998) might depend on the relationship of these repeats to transposable elements (Flavell et al., 1994; Jurka, 1998). Indirect evidence for activation of dormant transposons comes from a study of hybridization between Nicotiana otophora and N. tabacum in which an otophora chromosome segment containing a heterochromatic knob and a ower color gene became spontaneously unstable (Burns and Gerstel, 1967; Gerstel and Burns, 1967). Instability was evident as chromosome breakage as well as heterochomatin expansion at the knob site (Figure 5). Knob expansion could double the size of the affected abnormal chromosomes. In some cells the abnormal chromosomes

Figure 5. Expansion of chromosomes in Nicotiana allopolyploids. A. The images are from microphotographs of Gerstel and Burns (1967). Normal chromosomes (some of the largest in the normal karyotype), abnormal chromosomes, and megachromosomes are shown to the same scale. B. The progressive expansion of two normal chromosomes to abnormal and mega length was attributed to a single heterochromatic region, presumably a knob, of Nicotiana otophora (N.o.) that had been transferred by recombination to a Nicotiana tabacum (N.t.) chromosome. Chromosome expansion was accompanied by ower color variegation induced by chromosomes breakage followed by loss of the heterochromatic block and of the knob-distal Cov gene (Burns and Gerstel, 1967).

underwent an even more dramatic change: they increased their length by 20- to 30-fold to become megachromosomes! Cells that harbored either the abnormal chromosomes or the megachromosomes exhibited condensed chromatin regions in interphase nuclei that resembled a Barr body, the inactivated X-chromosome of female mammalian cells. This indicated that the additional DNA in these chromosomes was packaged as heterochromatin. Megachromosomecontaining cells were always surrounded by cells with the simple abnormal chromosomes, suggesting that the formation of the megachromosomes occurred in a single cell division cycle and that their presence blocked further cell divisions. In conclusion, these rarely cited studies document a remarkable case of heterochromatin expansion and instability in allopolyploid hybrids. While these studies on chromosome expansion in Nicotiana predated modern molecular analysis, they bear a striking similarity to those made in wallabie hybrids (ONeill et al., 1998). In these marsupials,

[ 271 ]

392 chromosome expansion was caused by the runaway replication of a retrotransposon which, in turn, was associated with a sharp decline of cytosine DNA methylation (discussed below). Therefore, hybridization of Nicotiana allopolyploids may have reactivated dormant transposons. A. thaliana and Cardaminopsis arenosa (Kamm et al., 1995; OKane et al., 1996). If the types of genes sampled by induced mutagenesis are representative of all genes, the level of genetic inactivation inferred from mutagenesis experiments appears higher than one might expect from experiments that monitored the expression or structure of sampled homeologous genes (see above). There are two possible explanations that may reconcile these data. First, inactivation may stem from alterations not detected by the assay used. For example, missense mutations could not alter transcription of the affected gene. Second, the high induced-mutation rate may have an epigenetic base, resulting from the formation of epialleles, which are manifested phenotypically but without changes in DNA sequence (Jacobsen and Meyerowitz, 1997). Analysis of gene expression in synthetic allopolyploids In synthetic allopolyploid hybrids genomic restructuring and unstable phenotypes could stem from alterations in gene regulation (Matzke et al., 1999). Perhaps the most dramatic, but sparsely investigated phenotypic symptoms of instability are sterility and lethality. Most synthetic allopolyploids display poor development and reduced survival of gametophytes and following fertilization, high lethality among the zygotes, usually during embryonic development. In crosses between Nicotiana species extensive lethality is also seen at the seedling stage (Marubashi et al., 1999). Allopolyploids that survive are usually vigorous, yet they do show some unusual characteristics such as homeotic phenotypes in Digitalis (Schwanitz, 1957), Gilia (Grant, 1956) and cotton (Meyer, 1970), ower variegation and tumor formation in Nicotiana (Kehr and Smith, 1954; Burns and Gerstel, 1967), and dominance of the hybrid phenotype by one parent in triticale (Heslop-Harrison, 1990). Polyploidy alone can affect the expression of certain genes. For example, in yeast expression of several genes is affected by ploidy level, and the suppression of certain G1 cyclins is thought to be responsible for the increase in cell size that accompanies polyploidy (Galitski et al., 1999). In maize, the ploidy level can either increase or decrease expression, depending on the particular gene (Guo et al., 1996). In Arabidopsis, a case of transgene silencing is correlated with tetraploidy (Mittelsten Scheid et al., 1996). Much less is known about the effects of allopolyploidy

Phenotypic and molecular alterations Gene inactivation The redundancy of genetic information in polyploids implies that unless duplicated loci gain a useful function, they should be inactivated by mutations (Ohno, 1970). Gene inactivation is used here to dene loss of gene activity by genetic changes, wheras silencing refers to loss of gene activity by epigenetic changes (see below). What fraction of the genes is inactivated in natural allopolyploids? Most work relevant to inactivation involved selected genes that were studied by isozyme or mRNA analyses, or by DNA sequencing. The results suggest that inactivation, while demonstrable in some cases, is relatively infrequent (Hart, 1983; Rieseberg and Soltis, 1989; Vaucheret et al., 1989; Pichersky et al., 1990; Gastony, 1991; van Buuren et al., 1992; Soltis and Soltis, 1993; Song and Osborn, 1994; Gutierrez et al., 1994; Hanfstingl et al., 1994; DHont et al., 1994; Boisselier Dubayle et al., 1996; Odrzykoski et al., 1996; Henikoff and Comai, 1998a; Cronn et al., 1999; Small et al., 1999). An alternative approach that samples more genes is the comparative mutagenesis of an allopolyploid and its diploid parents. Recessive nulli- or hypomorphic mutations affecting phenotype can only occur at non-redundant loci, implying a lack of orthologous function in a homeologous gene. The ratio of the induced mutation rate for the allopolyploid to the rate for its parents can be used to measure gene inactivation at the sampled loci. Therefore, if allopolyploid and parents show the same induced mutation rate, inactivation of the redundant genes would be taken to be 100% (implying no gene redundancy in the allopolyploid). The frequency of mutations that reduce or abolish chlorophyll accumulation in diploid and allotetraploid wheat suggested that 20% of the duplicate genes in the allotetraploid were inactivated (Stadler, 1929; Mac Key, 1958). By scoring embryo lethal mutations representing about 500 loci (Meinke, 1994), I observed similar rates of induced mutations in the allotetraploid Arabidopsis suecica (unpublished results), a natural hybrid of

[ 272 ]

393 on gene regulation. Whereas synthetic autopolyploids are less stable than the corresponding diploids, they appear more stable than synthetic allopolyploids (Allard, 1960). For example, spontaneous and induced autopolyploids of A. thaliana are relatively stable and fertile (Scott et al., 1998), whereas synthetic allotetraploids are unstable and show poor fertility (Comai et al., 2000). Furthermore, diploid, triploid, tetraploid and pentaploid hybrids in Nicotiana all form genetic tumors with comparable frequency (Kehr and Smith, 1954). Therefore, allopolyploidy may have an even greater effect on gene regulation, probably because the incompatibilities between different genomes are more disruptive than changes caused by additional copies of the same genome. The genomic resources available in A. thaliana should facilitate the molecular analysis of gene regulation in allopolyploids. Thus, we synthesized allotetraploid hybrids of A. thaliana and C. arenosa, which are the synthetic counterpart of the natural allotetraploid A. suecica. The synthetic allotetraploids had diploid sets of the parental genomes as indicated by the presence of 26 chromosomes, 10 from A. thaliana and 16 from C. arenosa, and by molecular marker analysis (Comai et al., 2000). In addition to displaying frequent sterility, embryo lethality and great variability in many morphological features, some hybrids (5 to 80% depending on the trait) displayed unusual phenotypes including homeotic aberrations and chimeric ower morphology. We compared gene expression in the hybrids and in the parents. To identify changes due to allopolyploidization, but not differences in ploidy (Galitski et al., 1999; Guo et al., 1996), we used lines with the same ploidy: the parents were autotetraploid and the progeny were allotetraploid. We employed AFLP-cDNA analysis, a PCR-based method that displays random restriction enzyme fragments of cDNA on denaturing polyacrylamide gels (Bachem et al., 1996). The analyses revealed several products that were absent in the F2 hybrids, although they were present in the parents and in a mixture of parental mRNAs. By RT-PCR analyses we conrmed silencing of three genes out of about 20 silencing candidate genes examined. WU-BLASTN similarity analyses (W. Gish, 19961999, http://blast.wustl.edu) and Southern blotting revealed that these three genes contain repetitive DNA. In one case, the repetitive element was determined to be a solo long terminal repeat, an LTR found alone and not anking an intact retrotransposon (Wirth et al., 1983). Interestingly, comparison of cytosine methylation in these families of LTRs by HpaII and MspI restriction digestion revealed strong CG methylation in both hybrids and parents, but decreased methylation at CNG sites in the hybrids. These results suggest that a thorough comparison of gene regulation in allopolyploids and their parents should help in understanding the consequences of hybridization on genomic function. Furthermore, they indicate that rapid gene silencing occurs in synthetic allopolyploids and point to a relationship between the silenced genes and repetitive DNA of presumed heterochromatic origin. Effect of DNA cytosine demethylation To further explore the link between instability in allotetraploids and cytosine DNA methylation, we investigated the effect of 5-aza-2 -deoxycytidine (azaC), an inhibitor of cytosine DNA methylases. Since azaC derepresses silenced genes (Hepburn et al., 1983; Bender and Fink, 1995), one might expect that azaCtreated synthetic allopolyploids would show fewer abnormalities. The results, however, showed otherwise. Whereas tetraploid parents and ve diploid A. thaliana ecotypes treated with azaC grew normally, the synthetic hybrids produced abnormal phenotypes at high frequency (Comai, unpublished observations). The abnormal phenotypes included dwarsm, aberrant branching patterns, fasciation, homeotic phenotypes and tumors suggesting that certain genes regulating development were malfunctioning. In contrast, control synthetic hybrids displayed the usual variability but failed to show the high frequency and intensity of aberrant phenotypes displayed by the azaC-treated cohort. These results show that the synthetic hybrids were phenotypically hypersensitive to demethylation. Thus, the increased rate of abnormalities upon demethylation suggests that this phenotypic instability resulted, directly or indirectly, from demethylation, and perhaps derepression, of certain DNA sequences. Nucleolar dominance Nucleolar dominance refers to the silencing in hybrids of one parental set of rRNA genes. A. suecica, the natural allopolyploid, displays silencing of the A. thaliana rRNA genes. Investigation of nucleolar dominance in the same synthetic hybrids that were used for gene silencing studies (see above) revealed that the F1 hybrids differed from one another in rRNA gene expression, showing variation between co-dominance and dominance of the C. arenosa rRNA

[ 273 ]

394 protein or by interacting proteins. As a result of these constraints, interactive components of a system evolve as a coordinated set. Upon speciation, orthologous sets may diverge from each other as exemplied in Figure 6. Upon hybridization, poorly matched components would be forced to interact and may function aberrantly. For example, expression of a diverged mutS allele caused failure of mismatch repair in bacteria (Prudhomme et al., 1991). Similarly, even slight alterations in the structure of the protein subunits of complexes involved in chromatin regulation should result in altered function. Deciency in the maintenance of DNA methylation could have severe consequences such as the derepression of dormant tranposons (see below). Epigenetic model Epigenetics refers to heritable changes in phenotype, and hence in gene regulation, that are not caused by changes in DNA sequence. One epigenetic phenomenon, homology-dependent gene silencing (HDGS), involves the suppression of one gene by another homologous gene (Wolffe and Matzke, 1999), and it is reviewed elsewhere in this issue. What features of the genomic and phenotypic instabilities of synthetic allopolyploids implicate epigenetic phenomena? Some genomic alterations suggest the derepression of transposons that were dormant in the parents (Gerstel and Burns, 1967). The abnormal phenotypes of the allopolyploids are better explained as being caused by epigenetic changes than by mutation (Grant, 1956; Schwanitz, 1957; Meyer, 1970; Comai, 2000). Genes that were rapidly silenced upon allopolyploidization were related to repetitive DNA elements (Comai, 2000), a common characteristic of genes susceptible to HDGS. The allopolyploids were phenotypically hypersensitive to genome demethylation suggesting a relationship between chromatin suppression of genes, which requires methylation (Jeddeloh et al., 1998; Wolffe and Matzke, 1999), and the variable hybrid phenotype (Comai, 2000). Taken together these observations indicate that allopolyploid formation may be associated with epigenetic changes. Epigenetic interactions in the hybrids may occur between homeologous genes or between genes and related sequences within heterochromatin. Epigenetic interactions between homeologous genes Genes that have silencing properties are usually associated with repeats: a type of silencing called paramu-

Figure 6. Assembly of a defective heteromeric protein complex in a hybrid. Subunits of a protein complex vary slightly between species. In the hybrid, a complex assembled from incompatible subunits may function irregularly or not at all.

genes (Chen et al., 1998). This instability was resolved in the F2 plants which consistently showed silencing of the A. thaliana genes and dominance of the C. arenosa rRNA genes. This dominance relationship could, however, be reversed by changing the parental genomic ratio from 1:1 (AACC) to 3:1 (AAAC; A and C represent the haploid genome of A. thaliana and C. arenosa, respectively). Stochastic onset of nucleolar dominance and a reversal of the direction by chromosomal dosage are difcult to explain with the normal rules of genetics. Instead, an epigenetic interaction between the ribosomal RNA genes of the two parents may determine the outcome.

Mechanisms generating instabilities in synthetic allopolyploids Genome instability and gene silencing in allopolyploids may stem from impairment of mechanisms regulating important genomic functions, such as the maintenance and remodeling of chromatin states. Two general models, one genetic and the other epigenetic, can be proposed to explain instability and silencing. The events predicted by the models could occur sequentially or independently. Both models extend a proposal by Dobzhansky (1937) to explain hybrid inviability. According to Dobzhansky, while certain genes are neutral or advantageous in their species of origin, they are deleterious in a hybrid genetic background due to the accumulation of incompatible features since divergence of the two taxa. Genetic model This model of instability is based upon the mutual evolutionary constraint exerted by subunits of the same

[ 274 ]

395 tation results from the interaction of incompatible alleles which often contain transposon-related sequences and repeats (Brink et al., 1968; Hollick et al., 1997; Wolffe and Matzke, 1999). Within a species, selection should act to avoid allelic divergences that raise the silencing potential beyond a tolerable threshold. On the other hand, homeologous genes sequestered in different diploid species should be free to diverge and accumulate characteristics that would emerge as incompatible when the homeologous genes are united by hybridization (Henikoff and Comai, 1998b). Thus, the joining of homeologous genomes might bring together genes that undergo silencing interactions. Epigenetic interactions between a gene and related heterochromatic repeats Interference of transposons with the regulation of euchromatic genes was rst described by McClintock (1965). Since the suggestion that HDGS arose as a mechanism to silence transposons (and viruses) has gained acceptance (Matzke et al., 1966; Bestor, 1990; Flavell, 1994; Martienssen, 1998a; Matzke and Matzke, 1998b; Baulcombe, 1999), the frequent presence of transposons near euchromatic genes has spawned the theory that gene silencing is the effect of an epigenetic mechanism to suppress transposons (Martienssen, 1998a; Matzke and Matzke, 1998a). The effect of a transposon insertion within a gene is not always predictable. In certain cases methylation of the transposon favors expression of that gene (by blocking production of transposon transcripts that interfere with the normal gene transcript), while demethylation of the transposon decreases expression of the gene (Martienssen, 1998b). However, methylation of a transcriptional regulatory region is likely to suppress transcription (Wolffe and Matzke, 1999). As a result, genes that have received transposon sequences that confer new regulatory properties (Wessler et al., 1995) should be highly sensitive to suppressive methylation of the transposonderived regulatory element. Extending McClintocks proposal that transposons may become activated in newly formed allopolyploids (McClintock, 1984), I suggest that it is the plant defense response against activated heterochromatic transposons that is responsible for silencing euchromatic genes that contain transposon-like sequences (Figure 7A). Once HDGS has been triggered by elevated expression of transposons, all sequences similar to those of the inducing transcripts would be targeted. The proposed silencing interaction between derepressed repeats and euchromatic genes provides an explanation for the mysterious homeotic phenotypes caused by genome-wide demethylation in Arabidopsis (Finnegan et al., 1996; Ronemus et al., 1996). One such phenotype was caused by a silenced gene, SUPERMAN, which was hypermethylated in its promoter region as if targeted by HDGS (Jacobsen and Meyerowitz, 1997). It is possible that the loss of methylation derepressed heterochromatic transposons containing sequences similar to those within the SUP gene promoter, initiating the events modeled in Figure 7A. The phenotypic susceptibility of allopolyploid hybrids, but not their parents, to the demethylating agent azaC might originate from a reduced ability of the hybrids to maintain suppressive chromatin complexes (see Figure 7B).

Conclusion Conventional genetic rules alone are inadequate to explain the instability of allopolyploids. Instead, epigenetic interactions between the parental genomes are likely to inuence the outcome of this type of hybridization by affecting both genome structure and function. Specically, the suppression of transposons that constitute a large fraction of plant genomes may be compromised in recent allopolyploids. The resulting trauma (McClintocks genomic shock) may lead to widespread perturbation of gene expression, perhaps by a silencing interaction between activated transposons and euchromatic genes. Unusual phenotypes may be readily observed in allopolyploids because they produce viable progeny whereas diploid hybrids between distantly related parents are sterile. Thus, the epigenetic interactions triggered by hybridization in allopolyploids would not be fundamentally different from those occurring in diploid hybrids. Qualitatively similar responses, of lesser intensity, may occur in intraspecic hybrids. Therefore, insight into genome function gained from the study of allopolyploidy may be applicable to hybrids of any type and may even elucidate positive interactions, such as those responsible for hybrid vigor.

Acknowledgments I would like to thank Arnold Bendich and Renata Fava-Ditt for suggestions and comments. The research

[ 275 ]

396

Figure 7. Model for silencing of genes by activated heterochromatic repeats. A. I, a well expressed gene contains a sequence similar to that found in repressed heterochromatic repeats; II, the repeats are activated by a genomic shock leading to production of RNA (and, possibly, transposition); III, expression of the repeats triggers a genome defense that leads to silencing of the repeats, but also to silencing of the cognate gene. Although the illustration depicts a case of transcriptional silencing by repressive chromatin, post-transcriptional silencing could also be involved. The gene sequence similar to the repeats could be located in the gene promoter, or enhancer, or transcribed region. B. Model to explain the hypersensitivity of Arabidopsis allopolyploids to 5-aza-2 -deoxycytidine (azaC) and the resistance of the parents. Allopolyploidization and azaC treatment are hypothesized to be incrementally effective in derepressing heterochromatic repeats. AzaC treatment would result in complete derepression in the hybrids, whose repeats are postulated to be already partially derepressed, but not in the parents, whose repeats are completely suppressed. Note that allopolyplody may, at times, cause complete derepression as illustrated in A. Additionally, the production in diploid Arabidopsis of homeotic phenotypes upon suppression of methylation by genetic means (Finnegan et al., 1996; Kakutani et al., 1996; Ronemus et al., 1996), but not by azaC treatement (Comai, unpublished results), suggests that the latter methods are more effective than azaC treatment.

in my laboratory on gene regulation in allopolyploids is supported by USDA-NRICGP Grant 9901352.

References
Allard, R.W. 1960. Principles of Plant Breeding, Wiley, New York, 485 pp. Allard, R.W., Garcia, P., Saenz-de-Miera, L.E. and Perez de la Vega, M. 1993. Evolution of multilocus genetic structure in Avena hirtula and Avena barbata. Genetics 135: 11251139.

Aragon-Alcaide, L., Reader, S., Miller, T. and Moore, G. 1997. Centromeric behaviour in wheat with high and low homoeologous chromosomal pairing. Chromosoma 106: 327333. Bachem, C.W.B. et al. 1996. Visualization of differential gene expression using a novel method of RNA ngerprinting based on AFLP: analysis of gene expression during potato tuber development. Plant J. 9: 745753. Bailis, A.M. and Rothstein, R. 1990. A defect in mismatch repair in Saccharomyces cerevisiae stimulates ectopic recombination between homeologous genes by an excision repair dependent process. Genetics 126: 535547. Baulcombe, D. 1999. Viruses and gene silencing in plants. Arch. Virol. Suppl. 15: 189201.

[ 276 ]

397
Bender, J. and Fink, G.R. 1995. Epigenetic control of an endogenous gene family is revealed by a novel blue uorescent mutant of Arabidopsis. Cell 83: 725734. Bestor, T.H. 1990. DNA methylation: evolution of a bacterial immune function into a regulator of gene expression and genome structure in higher eukaryotes. Phil. Trans. R. Soc. Lond. B Biol. Sci. 326: 179187. Boisselier Dubayle, M.C., Lambourdire, J. and Bischler, H. 1996. Progeny analysis by isozyme markers in the polyploid liverwort Plagiochasma rupestre. Can. J. Bot. 74: 521527. Brink, R.A., Styles, E.D. and Axtell, J.D. 1968. Paramutation: directed genetic change. Paramutation occurs in somatic cells and heritably alters the functional state of a locus. Science 159: 161170. Buckler, E.S. et al. 1999. Meiotic drive of chromosomal knobs reshaped the maize genome. Genetics 153: 415426. Burns, J. and Gerstel, D. 1967. Flower color variegation and instability of a block of heterochromatin in Nicotiana. Genetics 57: 155167. Chambers, S.R., Hunter, N., Louis, E.J. and Borts, R.H., 1996. The mismatch repair system reduces meiotic homeologous recombination and stimulates recombination-dependent chromosome loss. Mol. Cell Biol. 16: 61106120. Chen, Z.J., Comai, L. and Pikaard, C.S. 1998. Gene dosage and stochastic effects determine the severity and direction of uniparental ribosomal RNA gene silencing (nucleolar dominance) in Arabidopsis allopolyploids. Proc. Natl. Acad. Sci. USA 95: 1489114896. Clausen, R. and Goodspeed, T. 1925. Interspecic hybridization in Nicotiana. II. A tetraploid glutinosa-tabacum hybrid, an experimental verication of the Winges hypothesis. Univ. Calif. Pub. Bot. 11: 245256. Claverys, J.P., Mejean, V., Gasc, A.M. and Sicard, A.M., 1983. Mismatch repair in Streptococcus pneumoniae: relationship between base mismatches and transformation efciencies. Proc. Natl. Acad. Sci. USA 80: 59565960. Comai, L., Tyagi, A.P., Winter, K., Holmes-Davis, R., Reynolds, S., Stevens, Y. and Byers, B. 2000. Phenotypic instability and rapid gene silencing in newly formed Arabidopsis allotetraploids. Plant Cell 12 (in press) Cronn, R.C., Small, R.L. and Wendel, J.F. 1999. Duplicated genes evolve independently after polyploid formation in cotton. Proc. Natl. Acad. Sci. USA 96: 1440614411. DHont, A. et al. 1994. A molecular approach to unraveling the genetics of sugarcane, a complex polyploid of the Andropogoneae tribe. Genome 37: 222230. Digby, L., 1912. The citology of Primula kewensis and of other related Primula hybrids. Ann. Bot. 26: 357388. Dobzhansky, T. 1937. Genetics and the Origin of Species. Columbia University Press, New York. Endo, T.R. 1990. Gametocidal chromosomes and their induction of chromosome mutations in wheat. Jpn. J. Genet. 65: 135152. Feldman, M. et al. 1997. Rapid elimination of low-copy DNA sequences in polyploid wheat: a possible mechanism for differentiation of homoeologous chromosomes. Genetics 147: 13811387. Finnegan, E.J., Peacock, W.J. and Dennis, E.S. 1996. Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development. Proc. Natl. Acad. Sci. USA 93: 84498454. Fishel, R. and Kolodner, R.D. 1995. Identication of mismatch repair genes and their role in the development of cancer. Curr. Opin. Genet. Dev. 5: 382395. Flavell, A.J., Pearce, S.R. and Kumar, A. 1994. Plant transposable elements and the genome. Curr. Opin. Genet. Dev. 4: 838844. Flavell, R.B. 1994. Inactivation of gene expression in plants as a consequence of specic sequence duplication. Proc. Natl. Acad. Sci. USA 91: 34903496. Galitski, T., Saldanha, A.J., Styles, C.A., Lander, E.S. and Fink, G.R. 1999. Ploidy regulation of gene expression. Science 285: 251254. Gastony, G.J. 1991. Gene silencing in a polyploid homosporous fern: paleopolyploidy revisited. Proc. Natl. Acad. Sci. USA 88: 16021605. Gerstel, D.U. and Burns, J.A. 1967. Phenotypic and chromosomal abnormalities associated with the introduction of heterochromatin from Nicotiana otophora into N. tabacum. Genetics 56: 483502. Grant, V. 1956. The genetic structure of races and species in Gilia. Adv. Genet. 8: 89107. Guo, M., Davis, D. and Birchler, J.A. 1996. Dosage effects on gene expression in a maize ploidy series. Genetics 142: 13491355. Gutierrez, J.F., Vaquero, F. and Vences, F.J. 1994. Allopolyploid vs. autopolyploid origins in the genus Lathyrus (Leguminosae). Heredity 73: 2940. Hanfstingl, U. et al. 1994. Haplotypic divergence coupled with lack of diversity at the Arabidopsis thaliana alcohol dehydrogenase locus: roles for both balancing and directional selection? Genetics 138: 811828. Hart, G.E. 1983. Genetics and evolution of multilocus isozymes in hexaploid wheat. In: Isozymes: Current Topics in Biological and Medical Research, Alan R. Liss, New York, pp. 365. Henikoff, S. and Comai, L. 1998a. A DNA methyltransferase homolog with a chromodomain exists in multiple polymorphic forms in Arabidopsis. Genetics 149: 307318. Henikoff, S. and Comai, L. 1998b. Trans-sensing effects: the ups and downs of being together. Cell 93: 329332. Hepburn, A.G., Clarke, L.E., Pearson, L. and White, J. 1983. The role of cytosine methylation in the control of nopaline synthase gene expression in a plant tumor. J. Mol. Appl. Genet. 2: 315 329. Heslop-Harrison, J.S. 1990. Gene expression and parental dominance in hybrid plants. Development Suppl. 1990: 2128. Hollick, J.B., Dorweiler, J.E. and Chandler, V.L. 1997. Paramutation and related allelic interactions. Trends Genet. 13: 302308. Humbert, O., Prudhomme, M., Hakenbeck, R., Dowson, C.G. and Claverys, J.P. 1995. Homeologous recombination and mismatch repair during transformation in Streptococcus pneumoniae: saturation of the Hex mismatch repair system. Proc. Natl. Acad. Sci. USA 92: 90529056. Hunter, N., Chambers, S.R., Louis, E.J. and Borts, R.H. 1996. The mismatch repair system contributes to meiotic sterility in an interspecic yeast hybrid. EMBO J. 15: 17261733. Jacobsen, S.E. and Meyerowitz, E.M. 1997. Hypermethylated SUPERMAN epigenetic alleles in Arabidopsis. Science 277: 11001103. Jeddeloh, J.A., Bender, J. and Richards, E.J. 1998. The DNA methylation locus DDM1 is required for maintenance of gene silencing in Arabidopsis. Genes Dev. 12: 17141725. Jiang, C., Wright, R.J., El-Zik, K.M. and Paterson, A.H. 1998. Polyploid formation created unique avenues for response to selection in Gossypium. Proc. Natl. Acad. Sci. USA 95: 44194424. Jiricny, J. 1998. Eukaryotic mismatch repair: an update. Mutat. Res. 409: 107121. Jurka, J. 1998. Repeats in genomic DNA: mining and meaning. Curr. Opin. Struct. Biol. 8: 333337. Kakutani, T., Jeddeloh, J.A., Flowers, S.K., Munakata, K. and Richards, E.J. 1996. Developmental abnormalities and epimu-

[ 277 ]

398
tations associated with DNA hypomethylation mutations. Proc. Natl. Acad. Sci. USA 93: 1240612411. Kamm, A., Galasso, I., Schmidt, T. and Heslop-Harrison, J.S. 1995. Analysis of a repetitive DNA family from Arabidopsis arenosa and relationships between Arabidopsis species. Plant Mol. Biol. 27: 853862. Karpechenko, G.D. 1927. Polyploid hybrids of Raphanus sativus Brassica oleracea L. Bull. Appl. Bot. 17: 305408. Kehr, A.E. and Smith, H.H. 1954. Genetic tumors in Nicotiana hybrids, Brookhaven National Laboratory Symposia, vol. 6, Brookhaven National Laboratory, Upton, NY, pp. 5578. Kolodner, R.D. and Marsischky, G.T. 1999. Eukaryotic DNA mismatch repair. Curr. Opin. Genet. Dev. 9: 8996. Leitch, I.J. and Bennett, M.D. 1997. Polyploidy in angiosperms. Trends Plant Sci. 2: 470476. Liu, B. et al. 1998. Rapid genomic changes in newly synthesized amphiploids of Triticum and Aegilops. I. Changes in low-copy noncoding DNA sequences. Genome 41: 272277. Luo, M.-C., Dubcosvky, J. and Dvorak, J. 1996. Recognition of homeology by the wheat Ph1 locus. Genetics 144: 11951203. Mac Key, J. 1958. Mutagenic response in Triticum at different levels of ploidy, First Int. Wheat Genetics Symposium, Public Press, Winnipeg, Canada, pp. 88. Martienssen, R. 1998a. Chromosomal imprinting in plants. Curr. Opin. Genet. Dev. 8: 240244. Martienssen, R. 1998b. Transposons, DNA methylation and gene control. Trends Genet. 14: 263264. Marubashi, W., Yamada, T. and Niwa, M. 1999. Apoptosis detected in hybrids between Nicotiana glutinosa and N. repanda expressing lethality. Planta 210: 168171. Matzke, M., Matzke, A. and Eggleston, W. 1966. Paramutation and transgene silencing: a common response to invasive DNA? Trends Plant Sci. 1: 382388. Matzke, M.A. and Matzke, A.J. 1998a. Epigenetic silencing of plant transgenes as a consequence of diverse cellular defence responses. Cell. Mol. Life Sci. 54: 94103. Matzke, M.A. and Matzke, A.J. 1998b. Gene silencing in plants: relevance for genome evolution and the acquisition of genomic methylation patterns. Novartis Found. Symp. 214: 168180. Matzke, M.A., Mittelsten Scheid, O. and Matzke, A.J. 1999. Rapid structural and epigenetic changes in polyploid and aneuploid genomes. Bioessays 21: 761767. McClintock, B. 1965. The control of gene action in maize, Brookhaven National Laboratory Symposia, vol. 18, Brookhaven National Laboratory ,Upton, NY, pp. 162184. McClintock, B. 1984. The signicance of responses of the genome to challenge. Science 226: 792801. Meinke, D.W. 1994. Seed development in Arabidopsis thaliana. In: E.M. Meyerowitz and C.R. Somerville (Eds.) Arabidopsis, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 253295. Meyer, V.G. 1970. A facultative gymnosperm from an interspecic cotton hybrid. Science 169: 886888. Mikhailova, E.I. et al. 1998. The effect of the wheat Ph1 locus on chromatin organisation and meiotic chromosome pairing analysed by genome painting. Chromosoma 107: 339350. Mittelsten Scheid, O., Jakovleva, L., Afsar, K., Maluszynska, J. and Paszkowski, J. 1996. A change of ploidy can modify epigenetic silencing. Proc. Natl. Acad. Sci. USA 93: 71147119. Modrich, P. and Lahue, R. 1996. Mismatch repair in replication delity, genetic recombination, and cancer biology. Annu. Rev. Biochem. 65: 101133. Moore, G. 1998. To pair or not to pair: chromosome pairing and evolution. Curr. Opin. Plant Biol. 1: 116122. OKane, S.R.J., Schaal, B.A. and Al-Shehbaz, I.A. 1996. The origins of Arabidopsis suecica (Brassicaceae) as indicated by nuclear rDNA sequences. Syste. Bot. 21: 559566. ONeill, R.J., ONeill, M.J. and Graves, J.A. 1998. Undermethylation associated with retroelement activation and chromosome remodelling in an interspecic mammalian hybrid. Nature 393: 6872. Odrzykoski, I.J., Chudzinska, E. and Szweykowski, J. 1996. The hybrid origin of the polyploid liverwort Pellia borealis. Genetica 98: 7586. Ohno, S. 1970. Evolution by Gene Duplication, Springer-Verlag, Heidelberg, Germany. Petit, M.A., Dimp, J., Radman, M. and Echols, H. 1991. Control of large chromosomal duplications in Escherichia coli by the mismatch repair system. Genetics 129: 327332. Pichersky, E., Soltis, D. and Soltis, P. 1990. Defective chlorophyll a/b-binding protein genes in the genome of a homosporous fern. Proc. Natl. Acad. Sci. USA 87: 195199. Prolla, T.A. 1998. DNA mismatch repair and cancer. Curr. Opin. Cell Biol. 10: 311316. Prudhomme, M., Mejean, V., Martin, B. and Claverys, J.P. 1991. Mismatch repair genes of Streptococcus pneumoniae: HexA confers a mutator phenotype in Escherichia coli by negative complementation. J. Bact. 173: 71967203. Ramsey, J. and Schemske, D.W. 1998. Pathways, mechanisms, and rates of polyploid formation in owering plants. Annu. Rev. Ecol. Syst. 19: 467501. Rayssiguier, C., Thaler, D.S. and Radman, M. 1989. The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants. Nature 342: 396401. Rhoades, M.M. 1942. Preferential segregation in maize. Genetics 27: 395407. Rieseberg, L.H. and Noyes, R.D. 1998. Genetic map-based studies of reticulate evolution in plants. Trends Plant Sci. 3: 254259. Rieseberg, L.H. and Soltis, D.E. 1989. Assessing the utility of isozyme number for determining ploidal level: evidence from Helianthus and Heliomeris (Asteraceae). Aliso 12: 277286. Ronemus, M.J., Galbiati, M., Ticknor, C., Chen, J. and Dellaporta, S.L. 1996. Demethylation-induced developmental pleiotropy in Arabidopsis. Science 273: 654657. Schwanitz, F. 1957. Spornbildung bei einem Bastard zwischen drei Digitalis-Arten. Biol. Zentralbl. 76: 226231. Scott, R.J., Spielman, M., Bailey, J. and Dickinson, H.G. 1998. Parent-of-origin effects on seed development in Arabidopsis thaliana. Development 125: 33293341. Sears, E.R. 1976. Genetic control of chromosome pairing in wheat. Annu. Rev. Genet. 10: 3151. Selva, E.M., New, L., Crouse, G.F. and Lahue, R.S. 1995. Mismatch correction acts as a barrier to homeologous recombination in Saccharomyces cerevisiae. Genetics 139: 11751188. Small, R.L., Ryburn, J.A. and Wendel, J.F. 1999. Low levels of nucleotide diversity at homoeologous Adh loci in allotetraploid cotton (Gossypium L.). Mol. Biol. Evol. 16: 491501. Soltis, D.E. and Soltis, P.S. 1993. Molecular data and the dynamic nature of polyploidy. Crit. Rev. Plant Sci. 12: 243273. Soltis, D.E. and Soltis, P.S. 1995. The dynamic nature of polyploid genomes. Proc. Natl. Acad. Sci. USA 92: 80898091. Song, K., Lu, P., Tang, K. and Osborn, T.C. 1995. Rapid genome change in synthetic polyploids of Brassica and its implications for polyploid evolution. Proc. Natl. Acad. Sci. USA 92: 7719 7723.

[ 278 ]

399
Song, K. and Osborn, T.C. 1994. A method for examining expression of homologous genes in plant polyploids. Plant Mol. Biol. 26: 10651071. Stadler, I.J. 1929. Chromosome number and the mutation rate in Avena and Triticum. Proc. Natl. Acad. Sci. USA 15: 876881. Ungerer, M.C., Baird, S.J., Pan, J. and Rieseberg, L.H. 1998. Rapid hybrid speciation in wild sunowers. Proc. Natl. Acad. Sci. USA 95: 1175711762. van Buuren, M., Neuhaus, J.M., Shinshi, H., Ryals, J. and Meins, F., Jr. 1992. The structure and regulation of homeologous tobacco endochitinase genes of Nicotiana sylvestris and N. tomentosiformis origin. Mol. Gen. Genet. 232: 460469. Vaucheret, H., Vincentz, M., Kronenberger, J., Caboche, M. and Rouze, P. 1989. Molecular cloning and characterisation of the two homologous genes coding for nitrate reductase in tobacco. Mol. Gen. Genet. 216: 1015. Vega, J.M. and Feldman, M. 1998a. Effect of the pairing gene Ph1 and premeiotic colchicine treatment on intra- and interchromosome pairing of isochromosomes in common wheat. Genetics 150: 11991208. Vega, J.M. and Feldman, M. 1998b. Effect of the pairing gene Ph1 on centromere misdivision in common wheat. Genetics 148: 12851294. Volkov, R.A., Borisjuk, N.V., Panchuk, II, Schweizer, D. and Hemleben, V. 1999. Elimination and rearrangement of parental rDNA in the allotetraploid Nicotiana tabacum. Mol. Biol. Evol. 16: 311320. Wendel, J.F., Schnabel, A. and Seelanan, T. 1995. Bidirectional interlocus concerted evolution following allopolyploid speciation in cotton (Gossypium). Proc. Natl. Acad. Sci. USA 92: 280284. Wessler, S.R., Bureau, T.E. and White, S.E. 1995. LTRretrotransposons and MITEs: important players in the evolution of plant genomes. Curr. Opin. Genet. Dev. 5: 814821. Whitkus, R., Doebley, J. and Lee, M. 1992. Comparative genome mapping of Sorghum and maize. Genetics 132: 11191130. Winge, O. 1917. The chromosomes, their number and general importance. C.R. Trav. Lab. Carlsberg 13: 131275. Wirth, T., Gloggler, K., Baumruker, T., Schmidt, M. and Horak, I. 1983. Family of middle repetitive DNA sequences in the mouse genome with structural features of solitary retroviral long terminal repeats. Proc. Natl. Acad. Sci. USA 80: 33273330. Wolffe, A.P. and Matzke, M.A. 1999. Epigenetics: regulation through repression. Science 286: 481486. Yu, H.G., Hiatt, E.N., Chan, A., Sweeney, M. and Dawe, R.K. 1997. Neocentromere-mediated chromosome movement in maize. J. Cell Biol. 139: 831840. Zhao, X.P. et al., 1998. Dispersed repetitive DNA has spread to new genomes since polyploid formation in cotton. Genome Res. 8: 479492.

[ 279 ]