Sequence elimination and cytosine methylation are rapid and reproducible responses of the genome to wide hybridization and allopolyploidy in wheat

Abstract

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.

Figure 1.

AFLP Fingerprints of Genomic DNAs in Diploid and Allopolyploid Wheat. The panel at left (EcoRI+AAC/MseI+CTA) included Ae. sharonensis (TH), Ae. umbellulata (TU), the F1 interspecific hybrid between TH and TU (F1), and the descendant allotetraploid (S1). The panel at right (EcoRI+ACC/MseI+CAG) included Ae. longissima (TL), T. urartu (TMU), the F1 hybrid, and the descendant allotetraploid (S1). Arrows indicate three different situations: disappearance of a band in only the allotetraploid plant (A); disappearance of a band in the F1 plant that was maintained in the allotetraploid plant (B); and a rare event in which a new band appeared in the F1 plant (C).

Figure 2.

DNA Gel Blot Hybridization with Three Different AIF Probes That Showed Band Disappearance with the AFLP Method. Genomic DNA of Ae. longissima (TL; genome S^lS^l), T. urartu (TMU; genome AA), and three independently made newly synthesized allotetraploids (S1 generation; genome S^lS^lAA) was digested and blotted on a nylon membrane. (A) AIF1 probe (Table 2) was isolated from T. urartu and hybridized to EcoRV-digested genomic DNA. Sequence elimination was found in both Ae. longissima (bottom arrow) and T. urartu (top arrow). (B) AIF2 probe (Table 2) was isolated from T. urartu and hybridized to BamHI-digested genomic DNA. This was the only probe that showed a single-copy fragment in both parents. The T. urartu band, shown by the arrow, disappeared in the allotetraploid. (C) AIF6 (Table 2) was isolated from T. urartu and hybridized to HindIII-digested genomic DNA. Two bands from T. urartu, shown by arrows, disappeared in the allotetraploid. This pattern of elimination also was shown with enzymes that are not sensitive to methylation, such as DraI, with all of the probes (data not shown).

Figure 3.

MSAP Patterns Detected in the Two Diploid Parents, the F1 Hybrid, and the Allotetraploid. The primer combination used was HM+TCAA/E+ACG. Lines 1 to 4 are Ae. sharonensis (TH), T. monococcum ssp aegilopoides (TMB), the F1 hybrid (F1), and the allotetraploid (S1), respectively. H and M refer to digestion with EcoRI+HpaII and EcoRI+MspI, respectively. Bands that showed alteration in methylation pattern in the F1 hybrid and/or the allotetraploid are shown by arrows.

Figure 4.

DNA Gel Blot Analysis Using MIF8 Probe That Showed an Alteration in MSAP Pattern in the Allotetraploid (S1) of the Cross between Ae. sharonensis (TH) and T. monococcum ssp aegilopoides (TMB). (A) Each DNA sample was digested with EcoRI+HpaII (H) and EcoRI+MspI (M), and the two digests were loaded on the gel side by side (HpaII digest on the left and MspI digest on the right). The arrow points to the fragment whose methylation is altered in the allotetraploid. (B) DNA samples were digested with DraI (not sensitive to cytosine methylation) using the same hybridization probe. MIF8 was obtained using primer combination HM+TCAA/E+ACT (Table 5).

Figure 5.

DNA Gel Blot Analysis of a Band Isolated from MSAP That Did Not Show Alteration in Methylation but Rather Showed Band Disappearance. The bands obtained by both isoschizomers in parent T. monococcum ssp aegilopoides (TMB) were eliminated in the first generation of the allotetraploid (S1). TH, Ae. sharonensis. (A) Each DNA sample was digested with EcoRI+HpaII (H) and EcoRI+MspI (M), and the two digests were loaded on the gel side by side (HpaII digest on the left and MspI digest on the right). (B) DNA samples were digested with EcoRV using the same hybridization probe. The probe was obtained using primer combination HM+TCAA/E+AAG. Arrows indicate the band of TMB that disappeared in the allotetraploid.