Interspecific or intergeneric hybridization, followed by chromosome doubling, can lead to the formation of new allopolyploid species. Recent studies indicate that allopolyploid formation is associated with genetic and epigenetic changes, although little is known about the type of changes that occur, how rapidly they occur, and the type of sequences involved. To address these matters, we have surveyed F1 hybrids between diploid species from the wheat (Aegilops and Triticum) group and their derived allotetraploids by screening a large number of loci using amplified fragment length polymorphism and DNA gel blot analysis and by assaying the extent of cytosine methylation. We found that sequence elimination is one of the major and immediate responses of the wheat genome to wide hybridization or allopolyploidy, that it affects a large fraction of the genome, and that it is reproducible. In one cross between Ae. sharonensis ؋ Ae. umbellulata , 14% of the loci from Ae. sharonensis were eliminated compared with only 0.5% from Ae. umbellulata, with most changes occurring in the F1 hybrid. In contrast, crosses between Ae. longissima ؋ T. urartu showed that sequence elimination was more frequent after chromosome doubling. Alterations in cytosine methylation occurred in ‫ف‬ 13% of the loci, either in the F1 hybrid or in the allopolyploid. For eight of nine bands that were isolated, the sequences that underwent elimination corresponded to low-copy DNA, whereas alterations in methylation patterns affected both repetitive DNA sequences, such as retrotransposons, and low-copy DNA in approximately equal proportions.

Interspecific or intergeneric hybridization, followed by chromosome doubling, can lead to the formation of new allopolyploid species. Recent studies indicate that allopolyploid formation is associated with genetic and epigenetic changes, although little is known about the type of changes that occur, how rapidly they occur, and the type of sequences involved. To address these matters, we have surveyed F1 hybrids between diploid species from the wheat (Aegilops and Triticum) group and their derived allotetraploids by screening a large number of loci using amplified fragment length polymorphism and DNA gel blot analysis and by assaying the extent of cytosine methylation. We found that sequence elimination is one of the major and immediate responses of the wheat genome to wide hybridization or allopolyploidy, that it affects a large fraction of the genome, and that it is reproducible. In one cross between AE: sharonensis x AE: umbellulata, 14% of the loci from AE: sharonensis were eliminated compared with only 0.5% from AE: umbellulata, with most changes occurring in the F1 hybrid. In contrast, crosses between AE: longissima x T. urartu showed that sequence elimination was more frequent after chromosome doubling. Alterations in cytosine methylation occurred in approximately 13% of the loci, either in the F1 hybrid or in the allopolyploid. For eight of nine bands that were isolated, the sequences that underwent elimination corresponded to low-copy DNA, whereas alterations in methylation patterns affected both repetitive DNA sequences, such as retrotransposons, and low-copy DNA in approximately equal proportions.

The genome of plants, like that of other eukaryotes, is organized into chromatin, a compact structure that reduces the accessibility of DNA to machineries such as transcription, replication, and DNA recombination and repair. Plant genes, which contain the characteristic ATPase/helicase motifs of the chromatin remodeling Swi2/Snf2 family of proteins, have been thoroughly studied, but their role in homologous recombination or DNA repair has received limited attention. We have searched for homologs of the yeast RAD54 gene, whose role in recombination and repair and in chromatin remodeling is well established. Forty Arabidopsis SWI2/SNF2 genes were identified and the function of a selected group of 14 was analyzed. Mutant analysis and/or RNAi-mediated silencing showed that 11 of the 14 genes tested played a role in response to DNA damage. Two of the 14 genes were involved in homologous recombination between inverted repeats. The putative ortholog of RAD54 and close homologs of ERCC6/ RAD26 were involved in DNA damage response, suggesting functional conservation across kingdoms. In addition, genes known for their role in development, such as PICKLE/GYMNOS and PIE1, or in silencing, such as DDM1, turned out to also be involved in DNA damage response. A comparison of ddm1 and met1 mutants suggests that DNA damage response is affected essentially by chromatin structure and that cytosine methylation is less critical. These results emphasize the broad involvement of the SWI2/SNF2 family, and thus of chromatin remodeling, in genome maintenance and the link between epigenetic and genetic processes.

Gene targeting, which is homologous recombination-mediated integration of an extra-chromosomal DNA segment into a chromosomal target sequence, enables the precise disruption or replacement of any gene. Despite its value as a molecular genetic tool, gene targeting remains an inefficient technology in most species. We report that expression of the yeast RAD54 gene, a member of the SWI2͞SNF2 chromatin remodeling gene family, enhances gene targeting in Arabidopsis by one to two orders of magnitude, from 10 ؊4 to 10 ؊3 in WT plants to 10 ؊2 to 10 ؊1 . We show that integration events, detected with an assay based on the use of a fluorescent seed marker, are precise and germinally transmitted. These findings suggest that chromatin remodeling is rate-limiting for gene targeting in plants and improves the prospects for using gene targeting for the precise modification of plant genomes.

The Saccharomyces cerevisiae RAD54 gene has critical roles in DNA double-strand break repair, homologous recombination, and gene targeting. Previous results show that the yeast gene enhances gene targeting when expressed in Arabidopsis thaliana. In this work we address the trans-species compatibility of Rad54 functions. We show that overexpression of yeast RAD54 in Arabidopsis enhances DNA damage resistance severalfold. Thus, the yeast gene is active in the Arabidopsis homologous-recombination repair system. Moreover, we have identified an A. thaliana ortholog of yeast RAD54, named AtRAD54. This gene, with close sequence similarity to RAD54, complements methylmethane sulfonate (MMS) sensitivity but not UV sensitivity or gene targeting defects of rad54D mutant yeast cells. Overexpression of AtRAD54 in Arabidopsis leads to enhanced resistance to DNA damage. This gene's assignment as a RAD54 ortholog is further supported by the interaction of AtRad54 with AtRad51 and the interactions between alien proteins (i.e., yeast Rad54 with AtRAD51 and yeast Rad51 with AtRad54) in a yeast two-hybrid experiment. These interactions hint at the molecular nature of this interkingdom complementation, although the stronger effect of the yeast Rad54 in plants than AtRad54 in yeast might be explained by an ability of the Rad54 protein to act alone, independently of its interaction with Rad51.