Rapid and Repeatable Elimination of a Parental Genome-Specific DNA Repeat (pGc1R-1a) in Newly Synthesized Wheat Allopolyploids

Recent work in the Triticum-Aegilops complex demonstrates that allopolyploidization is associated with an array of changes in low-copy coding and noncoding sequences. Nevertheless, the behavior and fate of repetitive DNA elements that constitute the bulk of nuclear DNA of these plant species is less clear following allopolyploidy. To gain further insight into the genomic events that accompany allopolyploid formation, we investigated fluorescence in situ hybridization (FISH) patterns of a parental-specific, tandem DNA repeat (pGc1R-1) on three sets of newly synthesized amphiploids with different parental species. It was found that drastic physical elimination of pGc1R-1 copies occurred in all three amphiploids in early generations. DNA gel-blot analysis confirmed the FISH data and estimates indicated that 70–90% of the copies of the pGc1R-1 repeat family were eliminated from the amphiploids by the second to third selfed generations. Thus, allopolyploidy in Triticum-Aegilops can be accompanied by rapid and extensive elimination of parental-specific repetitive DNA sequences, which presumably play a role in the initial stabilization of the nascent amphiploid plants. ALLOPOLYPLOIDS, derived from interspecific or copy, coding and noncoding DNA sequences (Feldman et al. 1997; Liu et al. 1998; Ozkan et al. 2001; Shaked intergeneric hybridizations, contain two or more divergent homeologous genomes. Following initial hyet al. 2001; Kashkush et al. 2002; Ma et al. 2004). Surprisingly, probably due to intrinsic difficulties in monitoring bridization and genome doubling, the newly formed amphiploid may undergo a one-step speciation process changes in only some members of a given repetitive DNA family, little attention has been paid to the behavthat can be a traumatic experience to the combined allopolyploid genomes (reviewed in Leitch and Benior and fate of this type of sequence, which constitutes the bulk of these plant genomes. Nonetheless, for the nett 1997; Matzke et al. 1999; Comai 2000; Wendel 2000; Rieseberg 2001; Adams and Wendel 2004; Feldfew relevant studies available, a generic finding is that reduction in copy number of some DNA repeats apman and Levy 2005). Indeed, a number of recent reports have documented rapid genetic and epigenetic peared to be associated with interspecific hybridization and allopolyploidy in Triticeae. For example, it was instability that often accompany nascent allopolyploidy (reviewed in Pikaard 2001; Levy and Feldman 2002, found that, on the basis of DNA gel-blot analysis, although the DNA repeat pAesKB52 hybridized to Aegilops 2004; Liu and Wendel 2002; Comai et al. 2003; LawtonRauh 2003; Osborn et al. 2003b; Chen et al. 2004; Madspeltoides, Ae. sharonensis, and Ae. longissima—three probable diploid progenitors to the B genome of various lung and Comai 2004; Soltis et al. 2004; Ma and Gustafson 2005). polyploid wheats—it did not hybridize to any of the polyploid wheat species tested, thus suggesting eliminaA series of studies on newly synthesized allopolyploids of Triticeae, and particularly of the Triticum-Aegilops tion and/or extensive sequence divergence of the DNA repeat since allopolyploid formation (Anamthawatcomplex, has been particularly revealing in detecting rapid genomic and epigenomic changes (Ozkan et al. Jonsson and Heslop-Harrison 1993). 2001; Shaked et al. 2001; Han et al. 2003, 2004; Ma et Moreover, significant reduction in copy numbers of al. 2004). Among the changes it was found that the most Spelt1, a repetitive subtelomeric DNA family that repretantalizing but still mysterious phenomenon was rapid, sents 2% of the Ae. speltoides genome, was found in reproducible, and often nonrandom elimination of lownatural tetraand hexaploid wheat, Triticum tugidum and T. aestivum, as well as in the first generations of amphiploids that have Ae. speltoides as a parent (Pest1Present address: Division of Biological Sciences, University of Missova et al. 1998; Salina et al. 2004). Similarly, a dissouri, Columbia, MO 65211. persed repetitive DNA family (pSp89.XI) that is Ae. Spel2Corresponding author: Laboratory of Molecular Epigenetics, Northtoides specific was also found to have a much reduced east Normal University, Renmin St. 5268, Changchun 130024, China. E-mail: baoliu6677@yahoo.com.cn abundance in tetraand hexaploid wheat (Daud and Genetics 170: 1239–1245 ( July 2005) D ow naded rom http/academ ic.p.com /genetics/a70/3/1239/6060413 by gest on 24 M arch 2022

A LLOPOLYPLOIDS, derived from interspecific or copy, coding and noncoding DNA sequences (Feldman et al. 1997;Liu et al. 1998;Ozkan et al. 2001; Shaked intergeneric hybridizations, contain two or more divergent homeologous genomes. Following initial hy-et al. 2001;Kashkush et al. 2002;Ma et al. 2004). Surprisingly, probably due to intrinsic difficulties in monitoring bridization and genome doubling, the newly formed amphiploid may undergo a one-step speciation process changes in only some members of a given repetitive DNA family, little attention has been paid to the behav-that can be a traumatic experience to the combined allopolyploid genomes (reviewed in Leitch and Ben-ior and fate of this type of sequence, which constitutes the bulk of these plant genomes. Nonetheless, for the nett 1997; Matzke et al. 1999;Comai 2000;Wendel 2000;Rieseberg 2001;Adams and Wendel 2004;Feld-few relevant studies available, a generic finding is that reduction in copy number of some DNA repeats ap-man and Levy 2005). Indeed, a number of recent reports have documented rapid genetic and epigenetic peared to be associated with interspecific hybridization and allopolyploidy in Triticeae. For example, it was instability that often accompany nascent allopolyploidy (reviewed in Pikaard 2001;Levy and Feldman 2002, found that, on the basis of DNA gel-blot analysis, although the DNA repeat pAesKB52 hybridized to Aegilops 2004; Liu and Wendel 2002;Comai et al. 2003;Lawton-Rauh 2003;Osborn et al. 2003b;Chen et al. 2004;Mad-speltoides, Ae. sharonensis, and Ae. longissima-three probable diploid progenitors to the B genome of various lung and Comai 2004;Soltis et al. 2004;Ma and Gustafson 2005).
polyploid wheats-it did not hybridize to any of the polyploid wheat species tested, thus suggesting elimina-A series of studies on newly synthesized allopolyploids of Triticeae, and particularly of the Triticum-Aegilops tion and/or extensive sequence divergence of the DNA repeat since allopolyploid formation (Anamthawat-complex, has been particularly revealing in detecting rapid genomic and epigenomic changes (Ozkan et al. Jonsson andHeslop-Harrison 1993). 2001;Shaked et al. 2001;Han et al. 2003Han et al. , 2004Ma et Moreover, significant reduction in copy numbers of al. 2004). Among the changes it was found that the most Spelt1, a repetitive subtelomeric DNA family that repretantalizing but still mysterious phenomenon was rapid, sents 2% of the Ae. speltoides genome, was found in reproducible, and often nonrandom elimination of lownatural tetra-and hexaploid wheat, Triticum tugidum and T. aestivum, as well as in the first generations of amphiploids that have Ae. speltoides as a parent (Pest- were not identified (Ozkan et al. 2003 suggested that the most parsimonious explanation for this sequence is lacking. In this study, we investigated behavior of pGc1R-1 in three sets of newly synthesized wheat allopolyploids RESULTS AND DISCUSSION (amphiploids) , each having as a Rapid elimination of pGc1R-1a-containing DNA separent one of the three Sitopsis species, Ae. speltoides, quences in three sets of synthetic wheat allopolyploids Ae. longissima, and Ae. sharonensis, that contain the DNA as revealed by FISH analysis: Clone pGc1R-1a is a 258repeat. We report that rapid elimination of pGc1R-1 bp fragment of a tandem repetitive element cloned from copies occurred in all three sets of synthetic amphiploids Ae. speltoides, which hybridizes, at the FISH level, to the irrespective of the difference in species combination telomeric and subtelomeric regions of most Ae. speltoides or ploidy levels. We discuss potential causes for this chromosomes (Friebe et al. 2000). This element also elimination of parental-specific DNA repeats and its poshybridizes, albeit to a lesser extent with regard to both sible role in the stabilization of newly formed allopolythe number of hybridization sites and the strength of ploid plants.
the FISH signal, to two other species of the diploid Aegilops Sitopsis section, Ae. longissima and Ae. sharo-

MATERIALS AND METHODS
nensis, but does not hybridize to Ae. searsii and Ae. bicornis (Zhang et al. 2002). Interestingly, although multiple  1987;Kerby et al. 1988;Dvorak and Zhang 1990;Jiang and Gill 1994;Badaeva et al. 1996;Daud and Gustafson 1996;Sasanuma et al. 1998;Wang et al. 2000), pGc1R-1a hybridizes to neither of the polyploid species (Friebe et al. 2000;Zhang et al. 2002; also Figure 1i). On the basis of this finding, it was suggested that this element was likely eliminated during and/or following the allopolyploidization event leading to speciation of the tetraploid wheat, T. turgidum (Zhang et al. 2002). Nevertheless, because T. turgidum is hundreds of thousands of years old (Feldman 2001), an equally plausible explanation is gradual sequence divergence after tetraploid formation. To distinguish these two possibilities, we investigated the FISH patterns of pGc1R-1 in three sets of newly synthesized amphiploids, each involving one of the three diploid species, Ae. longissima, Ae. sharonensis, and Ae. speltoides, as a parent. It was found that drastic loss of FISH signals (hybridization sites) occurred in all three sets of synthetic amphiploids (Figure 1), which indicates rapid elimination and strongly argues against gradual sequence divergence as a cause. Specifically, there were no chromosomes bearing a FISH site in T. urartu (accession TMU06, Figure 1a) and six (presum- where cells within a given synthetic amphiploid plant Only four chromosomes still bearing the FISH sites (arrows) were found largely heterogeneous with regard to the were detected. number of FISH sites. This discrepancy suggests that differential mechanisms may underlie the elimination of different DNA repeats under allopolyploidy condi-tions. Taken together, it is evident that repetitive DNA an independent approach, we performed DNA gel-blot analysis with pGc1R-1a as a probe on multiple enzyme sequences like pGc1R-1a and Spelt1 are extremely labile when the chromosomes are in a hybrid/allopolyploid digestions (see materials and methods). It was found state and undergo rapid physical elimination in the that, in all enzyme digests, the strength of the gel-blot initial stages following allopolyploidization. The elimihybridization signal of pGc1R-1a was drastically reduced nation appeared to accrue following selfed generations in all three sets of amphiploids compared with that of and was not dependent on a particular species combinathe corresponding pGc1R-1a-containing diploid parent tion and/or on ploidy levels, as in this study all three (Figure 2, a, c, and e; data not shown). Equal loading tested combinations at the tetraploid or octoploid levels and quality of the DNA samples were confirmed by showed a remarkably similar pattern and rate of eliminaprobing the same blots (after stripping off the pGc1Rtion. 1a signals) with three low-copy RFLP probes, PSR301, Validation of rapid pGc1R-1a elimination by DNA PSR593, and PSR743 (Gale et al. 1993), that are known gel-blot analysis: To validate the rapid elimination of to present in the plant lines (Figure 2, b, d, and f; pGc1R-1a in the three sets of synthetic amphiploids by data not shown). To have a rough estimation of the elimination in a quantitative manner, we took into consideration the different exposure time (2 hr for pGc1R-1a vs. 6 days for the two low-copy RFLP probes, respectively) during autoradiography, and the genome size difference between the amphiploids and their parents (assuming the basic null hypothesis for genomic additivity; Wendel 2000). Thus, taking these two factors into consideration and using the low-copy probes as a normalizing control, we estimated, by densitometric scanning of the gel blots, that ‫%08-07ف‬ of the pGc1R-1a family was eliminated from the two tetraploid amphiploids, Ae. longissima ϫ T. urartu and Ae. sharonensis ϫ T. monococcum, with Ͼ90% of the pGc1R-1a family being Figure 2.-DNA gel-blot analysis of elimination of pGc1R-1 copies in newly synthesized amphiploids, each with one of the three pGc1R-1-containing diploid Aegilops (section Sitopsis) species, Ae. longassima, Ae. sharonensis, and Ae. speltoides, as a parent. (a) Hybridization of pGc1R-1 to two individual plants of a three-generation-old (S3) amphiploid (allotetraploid) of Ae. longissima (accession TL05) ϫ T. urartu (accession TMU06) and its two parents. Enzymes used to digest the DNA are indicated. Significant loss of hybridization signal is evident in both amphiploid individuals as compared with the parent Ae. longissima (accession TL05). (b) Hybridization of a low-copy wheat RFLP marker (PSR301) to the same blots as used above after stripping off the hybridization signals. The near-equal strength of the hybridization signal provides a positive control for quantity, quality, and complete digestion of the genomic DNA used. (c) Hybridization of pGc1R-1 to two individual plants of a two-generation-old (S2) amphiploid (allotetraploid) of Ae. sharonensis (accession TH02) and T. monococcum (accession TMB02). Enzymes used to digest the DNA are indicated. Significant loss of hybridization signal is evident in both amphiploid individuals as compared with the parent Ae. sharonensis (accession TH02). (d) Hybridization of a low-copy wheat RFLP marker (PSR593) to the same blots used above after stripping off the hybridization signals (as in b). (e) Hybridization of pGc1R-1 to two individual plants of a one-generation-old (S1) amphiploid (octoploid) of T. aestivum (accession 252) and Ae. speltoides (accession 15-1). Enzymes used to digest the DNA are indicated. Significant loss of hybridization signal is evident in both amphiploid individuals as compared with the parental line, Ae. speltoides (accession 15-1). (f) Hybridization of a lowcopy wheat RFLP marker (PSR743) to the same blots used above after stripping off the hybridization signals (as in b and d). eliminated from the octoploid amphiploid of T. aesti-sequences may have played. It has been proposed that vum ϫ Ae. speltoides. Thus, the gel-blot results appeared a possible function for the nonrandom and rapid elimiin full agreement with the FISH data (Figure 1), and nation of the low-copy sequences and rendering them the combined evidence is more than compelling to as chromosome or genome specific in the resultant alloallow the conclusion that rapid and extensive eliminapolyploid is to accentuate the physical and/or genetic tion of pGc1R-1 copies occurred in all three sets of divergence between the homeologous chromosomes, newly synthesized amphiploids, each with an elementsuch that strict homologous meiotic paring (cytological containing Aegilops diploid species as a parent. diploidization) and reduced genetic redundancy (ge-Previous studies on another DNA repeat called netic diploidization) will be facilitated and hence may pAesKB52 whose 5Ј-region shares 98% sequence homolcontribute to allopolyploid speciation (Levy and Feldogy to pGc1R-1 (Anamthawat-Jonsson and Heslopman 2002; Feldman and Levy 2005; Ma and Gustafson Harrison 1993) showed that it hybridizes, at the gel-blot 2005). Although direct evidence for this proposition level, to Ae. speltoides, Ae. sharonensis, and Ae. longissima but is lacking, correlative evidence that showed a positive not to the natural tetra-and hexaploid wheats, T. turgicorrelation between the amount and rapidity of sedum and T. aestivum, hence implicating elimination of the quence elimination and the fertility of the various newly DNA repeat after allopolyploid formation (Anamthawatsynthesized wheat allopolyploids investigated appeared Jonsson and Heslop-Harrison 1993). Our data on the to corroborate this possibility ). On three sets of synthetic wheat amphiploids have not only the other hand, the elimination of a parental-specific confirmed the earlier speculation by providing solid DNA repeat in newly formed allopolyploids would by experimental evidence, but also showed that the elimino means have such a role; on the contrary, its eliminanation may occur rapidly, i.e., at the very initial stages tion will reduce the divergence of the parental genomes. following allopolyploidization.
Thus, we speculate that a possible role, if any, for the Comparison between elimination of low-copy and reelimination of parental-specific DNA repeats upon allopetitive DNA sequences with regard to possible causes polyploidy is to eliminate or mitigate the genomic inand roles following allopolyploidy in Triticeae: A series compatibility of parental genomes that would otherwise of previous studies have reported rapid elimination of be too divergent for harmonious coexistence and coorlow-copy, coding or noncoding sequences upon allodination. Moreover, it has been suggested that the purgpolyploidization in Triticeae (Feldman et al. 1997; ing of genetic incompatibility is essential for high fertilet al. 1998;Ozkan et al. 2001;Shaked et al. 2001;Kash-ity in a hybrid or allopolyploid with divergent parental kush et Ma et al. 2004). Although similar in genomes (Rieseberg 2001). Nevertheless, because of the phenomenology, we note that there are at least two lines proposed scarcity, if not absence, of homeologous recomof apparent difference between the elimination of lowbinations under allopolyploid conditions in the Aegilopscopy and repetitive sequences. The first difference lies Triticum complex (Feldman et al. 1997;Ozkan et al. 2001; in possible causes for the elimination of the two types Salina et al. 2004), the purging of unfavorable genic of DNA sequences. Probably due to technical reasons for interactions cannot be accomplished by Mendelian segdetection, the results of both this study and previously regation (Rieseberg 2001). Therefore, it is conceivable reported works (Daud and Gustafson 1996; Pestsova that the drastic elimination of the parental-specific, yet et al. 1998; Salina et al. 2004) on the elimination of dispensable, DNA repeats would lead to a more harmorepetitive DNA elements following allopolyploidization nious cellular environment and hence might improve are confined to genome-specific sequences, i.e., sefertility in newly formed wheat allopolyploids. It should quences present in only one of the parental species. In be noted, however, that compelling recent evidence has contrast, all eliminated low-copy sequences described so showed that homeologous recombinations do occur in far are present in both parental species, being rendered other (mainly dicotyledonous) plant allopolyploids (synchromosome or genome specific only after allopolythetic or natural), such as the Brassica complex (Osborn ploid formation (the so-called group II sequences in et al. 2003a;Osborn 2004;Pires et al. 2004;Udall et al. Feldman et al. 1997). Thus, the elimination or reduction 2005), Arabidopsis (Madlung et al. 2005), and Nicotiana of the DNA repeat hitherto described is obviously not . caused by incompatibility among divergent members of It is currently not clear whether the rapid elimination the specific repeat family originally residing in the two of DNA repeats that we observed represents a general, parental species and being brought together by allopolydirected event associated with allopolyploidy or merely ploidy, while incongruence of homology-dependent ina stochastic anomalous incident triggered by specific teraction between the parental copies has been thought parental combinations. Nonetheless, the fact that the to be a major cause for the elimination of the group II strikingly similar elimination events have repeatedly oclow-copy sequences (Liu and Wendel 2002; Feldman curred in all three sets of independently synthesized and Levy 2005).
amphiploids supports the former possibility. In addi-The second line of difference lies in a possible biological role, if any, that the elimination of the two types of tion, a recent study showed that elimination of parental-sequences sheds light on the phylogeny of the wheat B and G specific satellite DNA also repeatedly occurred in newly due to breakdown of normal repressive controls in the Friebe, B., L. L. Qi, S. Nasuda, P. Zhang, N. A. Tuleen et al., hybrids (Petrov et al. 1995;O'Neill et al. 1998