Introduction
Whole genome duplication (WGD) is widespread in eukaryotes , and has been linked to phenotypic diversification during evolution . The common ancestor of salmonids underwent a lineage-specific WGD event ~100 million years ago and a large proportion of the genome is retained in duplicate, offering a n ideal vertebrate system to understand the role of WGD in genome evolution. T he huge commercial importance of these species to aquaculture further demands im proved understanding of genome function and regulation, which is still poorly understood. In the current study, we make extensive use of the functional annotation data generated through the European AQUA-FAANG project , including 0.6 billion ATAC-Seq and 4 billion ChIP-Seq reads, to investigate duplicated regulatory elements in the genomes of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) . The objective was to examine the conservation of regulatory element activity through ontogeny in both species.
Methods
Duplicate-aware w hole genome alignments including Atlantic sal mon and r ainbow trout were generated with Cactus ( Armstrong et al, 2020) to align the duplicated syntenic regions in both species . The genome was broken into syntenic blocks before alignment ( Gundappa et al. 2022) . N orthern pike (Esox lucius ) was included in the alignments as a closely related outgroup to the salmonid-specific WGD . High-confidence ATAC-Seq peaks (open chromatin regions) representing multiple stages of embryogenesis, and six adult tissues at two stages of sexual maturation, were overlapped with the Cactus alignments. The coupling of sequence and regulatory element conservation in open chromatin regions was established with respect to duplicated and orthologous regions.
Results
Our alignments captured a high proportion of duplicated and orthologous sequences across the genomes of Atlantic salmon, rainbow trout and northern pike, validating the robustness of our approach. After cross-referencing open chromatin regions with these alignments, we split the data into distinct categories according to their conservation b etween the two salmonid species (Fig. 1). The proportion of open chromatin regions overlapping both duplicated sequences retained from WGD increased across embryogenesis, being highest at the late somitogenesis stage, and was variable across adult tissue types, with brain s howing the highest proportion (Fig. 1B). Reciprocally , we identified the lowest proportion of open chromatin regions in singleton sequences (i.e. where the other duplicated sequence was lost) at the equivalent stage of development and tissues (Fig. 1B).
Discussion
Our results validate the use of genome alignment to understand the dynamics of regulatory element activity across the duplicated genomes of salmonids. Our results are broadly consistent with the hourglass model of development (Duboule, 1994), suggesting highest evolutionary constraints on gene regulation during the phylotypic stage , a pattern previously observed across species, but not in relation to WGD. We are currently overlaying chromatin state annotations generated by ChromHMM to understand the co-evolution of specific regulatory element classes (e.g. promoters/enhancers) and duplicated gene expression across different stages and tissues in both salmonid species. In addition, we are linking the open chromatin regions to conserved non-coding elements of different evolutionary ages . The results of this work, by revealing conserved regulatory elements linked to salmonid phenotypes, will support the uptake of functional genomic information into salmonid genetics and selective breeding approaches supporting sustainable and profitable salmonid aquaculture.
Acknowledgement: The AQUA-FAANG project received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 817923 (www.aqua-faang.eu).
References
Armstrong, J., Hickey, G., Diekhans, M. , et al. (2020). Progressive Cactus is a multiple-genome aligner for the thousand-genome era . Nature, 587(7833), 246-251.
Duboule , D. (1994). Temporal colinearity and the phylotypic progression: a basis for the stability of a vertebrate Bauplan and the evolution of morphologies through heterochrony . Development, 1994 (Supplement), 135-142.
Gundappa, M. K., To, T. H., Grønvold, L., et al. (2022). Genome-Wide Reconstruction of Rediploidization Following Autopolyploidization across One Hundred Million Years of Salmonid Evolution . Mol Biol Evol, 39:msab310