Extensive and heritable epigenetic remodeling and genetic stability accompany allohexaploidization of wheat

Abstract

Allopolyploidy has played a prominent role in organismal evolution, particularly in angiosperms. Allohexaploidization is a critical step leading to the formation of common wheat as a new species, Triticum aestivum, as well as for bestowing its remarkable adaptability. A recent study documented that the initial stages of wheat allohexaploidization was associated with rampant genetic and epigenetic instabilities at genomic regions flanking a retrotransposon family named Veju. Although this finding is in line with the prevailing opinion of rapid genomic instability associated with nascent plant allopolyploidy, its relevance to speciation of T. aestivum remains unclear. Here, we show that genetic instability at genomic regions flanking the Veju, flanking a more abundant retroelement BARE-1, as well as at a large number of randomly sampled genomic loci, is all extremely rare or nonexistent in preselected individuals representing three sets of independently formed nascent allohexaploid wheat lines, which had a transgenerationally stable genomic constitution analogous to that of T. aestivum. In contrast, extensive and transgenerationally heritable repatterning of DNA methylation at all three kinds of genomic loci were reproducibly detected. Thus, our results suggest that rampant genetic instability associated with nascent allohexaploidization in wheat likely represents incidental and anomalous phenomena that are confined to by-product individuals inconsequential to the establishment of the newly formed plants toward speciation of T. aestivum; instead, extensive and heritable epigenetic remodeling coupled with preponderant genetic stability is generally associated with nascent wheat allohexaploidy, and therefore, more likely a contributory factor to the speciation event(s).

Figure 1

(A) Diagrams showing pedigree of three independently formed allohexaploid wheat lines, designated as Allo-960, Allo-AT5, and Allo-AT9, each with three to five selfed generations available for this study. (B) Typical chromosomal constitutions for the selected individuals from each of the three allohexaploid wheat lines, as revealed by multicolor GISH. All plant individuals used for this study are with transgenerational chromosomal stability at the subgenomic level (euploidy and lack of any intergenomic rearrangements), as shown in B, which is highly similar to the natural common wheat, T. aestivum. The pink-, green-, and blue-colored chromosomes are of the BB, AA, and DD genomes, respectively.

Figure 2

Genetic and DNA methylation changes in 11 studied individuals representing the three (S3–S5) or five (S1–S5) selfed generations of three independently formed allohexaploid wheat lines (Allo-960, Allo-AT5, and Allo-AT9). Two TD markers (Veju-TD and BARE-1–TD) and the AFLP marker were used to detect possible genetic changes, while their cytosine methylation-sensitive counterparts, two MSTD markers (Veju-MSTD and BARE-1–MSTD) and the MSAP marker, were used to detect possible alteration in cytosine methylation at the 5′-CCGG sites. The frequencies for the two major types of genetic changes, loss and gain, detected by the three kinds of markers, are depicted in A, while the frequencies of the four major patterns of cytosine methylation alteration as well as total, detected by Veju-MSTD, BARE-1–MSTD, and MSAP, respectively, are depicted in B–D. The detailed dataset for the scored variant bands is presented in Table S3 and Table S4.

Figure 3

Examples of typical MSAP and MSTD profiles for the same plants as in Figure 2 illustrating the four patterns of cytosine methylation alteration, CG hyper, CHG hyper, CG hypo, and CHG hypo, which can be unequivocally distinguished by these methylation-sensitive markers. A–F are produced by primer combinations of EcoRI-E + H/M-4, EcoRI-A + H/M-10, EcoRI-F + H/M-8, Veju + H/M-4, Veju + H/M-8, and BARE-1 + H/M-15, respectively.

Figure 4

The genomic bisulfite sequencing-based cytosine methylation maps and collective methylation levels (%) of the three types of cytosine residues, CG (red dots or columns), CHG (blue dots or columns), and CHH (green dots or columns), for each of the three variant MSAP loci [MSAP 19 (A), MSAP 27 (B), and MSAP 29 (C)] in three individual plants representing three successive selfed generations (S3–S5) of the nascent allohexaploid wheat line (AT9) and its parental species, T. turgidum, ssp. durum, cv. TTH01 and Ae. tauschii, line TQ27. The sequences for the studied loci are given in Figure S2, which are completely conserved between the two parental lines for AT9. For each analyzed locus, 15 clones were arbitrarily sequenced to reflect each biological sample. The calculated MPVs are included for comparison.