High-throughput Chromatin Conformation Capture (Hi-C) technologies can be used to investigate the three-dimensional genomic structure of plants. However, the practical utility of these technologies is impeded by significant background noise, hindering their capability in detecting fine 3D genomic structures. In this study, we optimized the Bridge Linker Hi-C technology (BL-Hi-C) to comprehensively investigate the 3D chromatin landscape of Brassica rapa and Brassica oleracea. The Bouquet configuration of both B. rapa and B. oleracea was elucidated through the construction of a 3D genome simulation. The optimized BL-Hi-C exhibited lower background noise compared to conventional Hi-C methods. Taking this advantage, we used BL-Hi-C to identify FLC gene loops in Arabidopsis, B. rapa, and B. oleracea. We observed that gene loops of FLC2 exhibited conservation across Arabidopsis, B. rapa, and B. oleracea. While gene loops of syntenic FLCs exhibited conservation across B. rapa and B. oleracea, variations in gene loops were evident among multiple paralogs FLCs within the same species. Collectively, our findings highlight the high sensitivity of optimized BL-Hi-C as a powerful tool for investigating the fine 3D genomic organization.

Whole genome doubling (WGD) triggers profound genomic and epigenetic reorganization, yet the functional dynamics of DNA methylation during this process remain incompletely resolved. Here, we integrate whole genome bisulfite sequencing (WGBS) and three-dimensional chromatin interaction data to display methylation landscapes in autotetraploid Arabidopsis thaliana. Our analysis reveals evolutionarily conserved spatial patterning of DNA methylation after WGD, with centromeric enrichment and telomeric depletion. Chromosome-level profiling identifies Chromosome 2 as the most highly methylated across CG, CHG, and CHH contexts, while Chromosome 1 shows the lowest methylation. Subcontext methylation analysis uncovers increases in methylation levels in autotetraploid Arabidopsis thaliana, most pronounced in the CHH context, yet global distribution patterns remain stable. Comparative methylation profiling around genes and transposable elements (TEs) reveals elevated CHH methylation in autotetraploid gene bodies and flanking regions, whereas TE bodies exhibit minimal changes despite minor flanking hypermethylation. Strikingly, 8% of chromatin compartments were restructured, and B-B interactions weakened in autotetraploid, while DNA methylation remained stable across shifting A/B compartments. Our findings suggest that DNA methylation serves as a resilient epigenetic modification during WGD, even if 3D chromatin architecture undergoes reorganization upon WGD in some degree.