The study isolated novel wheat repetitive elements using methylation-insensitive approaches. For the A genome (Triticum monococcum), the enzyme EcoO109I (recognition site: 5′-PuGGNCCPy-3′) was used to cleave genomic DNA irrespective of CpG methylation, enabling cloning of methylated repeats like retrotransposon-derived pTm6. For the D genome (Aegilops tauschii), methylation-insensitive MboI digestion combined with genomic subtraction isolated D-specific repeats (e.g., pAt1) independent of methylation status. The retroelement-like pTm58 exhibited a tRNA structure, a feature often linked to epigenetic regulation in noncoding RNAs. These methods effectively bypassed methylation barriers to access diverse repeats in wheat’s complex genome.

This study demonstrates that genome-wide DNA methylation repatterning, primarily CG hypermethylation, is a general and stable epigenetic response to nascent allohexaploidization in wheat. Unlike stochastic chromosomal/DNA changes confined to rare individuals, methylation alterations occurred pervasively (~11% of loci) and were transmitted stably across generations, affecting genic and repetitive regions. These findings highlight epigenetic remodeling as a fundamental mechanism for genome stabilization during wheat polyploid evolution.

Diverse transposable elements (TEs) fundamentally shape the epigenetic landscape of Aegilops tauschii by defining distinct chromatin states across TE orders/superfamilies and creating open chromatin regions, particularly within specific superfamilies like hAT-Ac near promoters, which associate with active histone marks (e.g., H3K9ac) and influence gene expression, highlighting their critical role in regulating the wheat D genome's epigenome.

Whole-genome doubling (WGD) in tetraploid Aegilops tauschii induced the enrichment of the repressive histone mark H3K27me3 specifically in the non-TE sequences flanking certain transposable elements (TEs) located within genic regions, particularly LINEs, CACTA, PIF/Harbinger, Tc1/Mariner, and unclassified DNA transposons, compared to the diploid progenitor. This increased H3K27me3 enrichment in the tetraploid line was negatively associated with the expression levels of the genes carrying these specific TEs, suggesting that these particular TE types may play a role in regulating ploidy-related gene expression by mediating differential deposition of H3K27me3 in their adjacent genic chromatin following WGD.

In wheat seedlings, specific JmjC histone demethylases (Tr-1A/1B/1D-JMJ2 and Tr-4B/7A-JMJ1 homologs) mediate drought response in roots by dynamically regulating H3K27me3 demethylation. Tr-1A/1B/1D-JMJ2 showed rapid upregulation at 1h post-PEG stress, activating early drought genes, while eight others (e.g., Tr-4B-JMJ1, Tr-7A-JMJ1) peaked at 24h, implicating them in late adaptation. Cis-elements (ABA, MYB, MBS) in their promoters further support epigenetic roles in drought resilience.

The study demonstrates that reduced chromatin accessibility in hexaploid wheat’s homologous chromosome arms underlies widespread gene suppression compared to its diploid progenitor, with DNA methylation differences playing only a minor role.

Diversiform transposons (TEs), constituting over 85% of the Aegilops tauschii genome, are the primary architects of its epigenetic landscape, accounting for ~92% of chromatin states. Different TE orders and superfamilies exhibit distinct chromatin state signatures. Crucially, specific TE superfamilies, particularly DNA/hAT-Ac, are significantly enriched in open chromatin regions, often near gene promoters and carrying transcription factor motifs. Active histone modifications (like H3K9ac) and activated chromatin states correlate strongly with these open regions, especially on distal TEs (e.g., LTR/Gypsy), suggesting their regulatory potential. TE-associated chromatin accessibility directly influences nearby gene expression. Thus, diverse TEs fundamentally structure the epigenetic framework, shaping chromatin states, accessibility, and gene regulation in the wheat D genome progenitor.