Matthew Peter Kent

Whole genome duplication (WGD) events during early vertebrate evolution may have promoted phenotypic innovation by expanding the genetic material for functional diversification. However, their ancient origins obscure how gene regulation evolved during rediploidization, the process that generates duplicate genes and regulatory elements following WGD. Salmonids provide a unique window to these mechanisms, because they experienced a more recent autotetraploid WGD and remain at an earlier stage of rediploidization. Here we generated 771 multiomics datasets spanning embryonic and adult tissues in Atlantic salmon and rainbow trout to investigate the evolution of gene expression and regulatory elements. Our analyses show that regulatory evolution following the salmonid WGD was shaped by developmental and tissue-specific constraints known to act across vertebrate species. We identify a peak of within-genome regulatory constraint at the vertebrate phylotypic period, associated with the functional retention of duplicated enhancer pairs harbouring deeply conserved non-coding elements enriched in motifs for master developmental transcription factors. We further identify roles for enhancers and transposable elements in the expression divergence of duplicated genes. This study advances the understanding of regulatory evolution following WGD and offers an open resource supporting vertebrate comparative genomics and research applications in salmonid aquaculture and conservation.

Reindeer and caribou (Rangifer tarandus) play key ecological, cultural, and economical roles across Arctic and Subarctic regions. However, genome-wide patterns of genetic diversity and population structure in semi-domesticated reindeer from Norway remain insufficiently characterized. Here we generated a high-quality chromosome-level genome assembly (GCA_965153185.1) for Norwegian semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus). The genome spans 2.54 Gb, with a scaffold N50 of 75.6 Mb, 96.1% complete Benchmarking Universal Single-Copy Orthologues, and 39,855 predicted protein-coding genes. Using this reference, we performed whole-genome resequencing of 175 individuals from five reindeer populations from Norway and 28 individuals from Iceland, identifying ~19.9 million genetic variants. Genetic differentiation was low among Norwegian populations (FST = 0.005 to 0.011), but substantially higher between Norway and Iceland (FST = 0.140 to 0.161). Populations from Norway exhibited similar levels of nucleotide diversity (π = 1.39 to 1.62 × 10−3), whereas the Icelandic population showed reduced diversity (π = 0.99 × 10−3) and approximately half as many segregating variants. Analyses of runs of homozygosity (ROH) and genome-wide heterozygosity indicated low levels of recent inbreeding across all populations, with long ROHs (>8 Mb) being rare. Despite reduced genetic diversity, the Icelandic population showed limited evidence of recent inbreeding. These patterns are consistent with contrasting demographic histories, with gene flow maintaining diversity among populations from Norway, while founder effects combined with long-term isolation have shaped the Icelandic population.

Understanding how to maintain and direct spermatogenesis in vitro is central to advancing reproductive biotechnologies in aquaculture species where the ability to generate gametes outside the organism could facilitate selective breeding, genetic modification, and germline preservation. However, current culture systems remain poorly defined in farmed fish species. In Atlantic salmon ( Salmo salar ), progress has been further limited by the absence of a comprehensive reference atlas of testicular cell types, making it difficult to determine how cells maintained in culture relate to their native counterparts. To address this, we first established a single-cell RNA atlas of the Atlantic salmon testis from freshly isolated tissue, resolving somatic and germ cell populations across all major stages of spermatogenesis. Primary testicular cells were then cultured under distinct conditions designed to promote either proliferation or differentiation for 14 days and subsequently subjected to single-cell RNA sequencing. To assign cell identities in cultured samples, the transcriptional profiles of cultured cells were computationally mapped onto the atlas, allowing direct comparison of cultured and native cell states. This approach revealed pronounced, condition-specific shifts in cellular composition. Proliferation medium supplemented with epidermal growth factors (EGF) and insulin-like growth factor (IGF) enriched spermatogonial populations, indicating preferential support of undifferentiated and actively dividing germ cells. In contrast, basal medium favoured the preferential survival of Sertoli cells in the absence of defined growth cues. A differentiation medium containing hormones that stimulate male gonad development (gonadotropins and androgens) failed to robustly promote meiotic progression. Further, comparative analysis of Sertoli cells across different conditions ( in vivo and in vitro ) revealed a loss of canonical identity markers and induction of stress-associated transcriptional programs in vitro compared to in vivo , indicating a shift away from specialised somatic function. Together, these findings establish the first single-cell reference atlas of Atlantic salmon testis and provide a framework for evaluating and optimising testis culture systems in salmonids. While early germ cell populations could be maintained and enriched in vitro, progression through later stages of spermatogenesis remained limited, indicating that important biological requirements of the native testicular environment are not yet fully recapitulated under current culture conditions.

Early embryogenesis is governed by tightly regulated transcriptional programs, including the maternal-to-zygotic transition (MZT), zygotic genome activation (ZGA), and maintenance of pluripotency. While these processes are well studied in model vertebrates, they remain poorly understood in salmonid fishes, whose genomes are shaped by a relatively recent whole genome duplication (WGD) event. Here, we present a temporally resolved transcriptomic analysis of Atlantic salmon (Salmo salar) embryogenesis using bulk RNA-seq across key stages spanning early embryogenesis. Dimensionality reduction and unsupervised clustering of gene expression revealed stage-specific transitions encompassing maternal RNA clearance, cell cycle regulation, and the onset of metabolic activity. We demonstrate that ZGA occurs early and in multiple phases, beginning soon after fertilization, accompanied by chromatin remodelling and the activation of epigenetic regulators. Duplicated gene pairs retained from the salmonid WGD frequently displayed asynchronous expression, indicative of functional divergence and the evolution of additional regulatory complexity of embryonic development. To gain insights into pluripotency, we integrated analyses of gene expression, transcription factor motifs, and chromatin accessibility, to reveal conserved regulators including genes encoding Pou5f3, Nanog, and Sox19b, alongside divergent functions of Klf family members. Our cross-stage profiling allowed us to define a novel panel of stably-expressed reference genes for normalization during quantitative PCR analyses, which were used to validate pluripotency- and differentiation-associated dynamics inferred by RNA-seq. Together, our findings delineate the transcriptional architecture of early embryogenesis in Atlantic salmon, revealing both conserved and lineage-specific features of pluripotency regulation, and providing a foundational resource for future functional genomics and stem cell applications in salmonids.

Genotype-to-phenotype (G2P) research in farmed animals has entered a phase in which deep genome annotation, multi-omics and AI-enabled prediction can nominate variants, regulatory mechanisms and cellular pathways at scale, but causal validation remains a major bottleneck. In vitro cellular systems, from tractable primary cells to organoids and other advanced models, provide experimentally controlled contexts in which massively parallel reporter assays and CRISPR-based perturbations can test variant effects and define cellular phenotypes. Here, we examine how in vitro approaches can be deployed across host–pathogen interactions, genotype-by-environment responses, nutrition, adaptation and One Health research, and outline priorities for integrating in vivo, in vitro and in silico data into mechanistic G2P workflows.

The Duroc-derived reference genome Sscrofa11.1 has provided a critical foundation for pig genomics, providing a high-quality reference genome for accurate variant detection and comparative genomics but does not capture breed-specific variation. Here, we present a near-complete, gap-free genome assembly for the Landrace pig (Landrace_v1, GCA_963921485.1), spanning all 20 chromosomes and totaling 2.6 Gb, including 176 Mb of sequence absent from Sscrofa11.1. Comparative analyses with recently published high-quality pig genomes reveal a conserved centromere organization across breeds, accompanied by substantial variation in repeat composition and length, and identify a pig specific pattern of telomere variant repeats across eight pig breeds. The improved resolution of repetitive regions in Landrace_v1 enables more complete reconstruction of complex gene families, including olfactory receptors, and uncovers structural variation at the KIT proto-oncogene receptor tyrosine kinase locus not represented in the Duroc reference. Together, these findings highlight the limitations of single-reference genomes and demonstrate the value of breed-specific assemblies for capturing genomic diversity and improving downstream analyses.

Early vertebrate autotetraploidization events may have enabled major innovations by expanding the genetic material for functional diversification, yet their ancient timing obscures how genome doubling reshaped gene regulatory evolution. Salmonids provide a unique window to these mechanisms, because they experienced a comparatively recent autotetraploidization and are earlier in the rediploidization process - which creates new genes and regulatory elements during evolution. Here, using large-scale multiomics spanning embryonic and adult tissues in two salmonids, we investigate gene regulatory evolution following genome doubling and rediploidization, which we show is governed by developmental and tissue-specific context, with a period of maximal constraint at advanced stages of embryogenesis. This work advances understanding of vertebrate genome evolution, while providing an open resource supporting salmonid aquaculture and conservation.

Norwegian Red (NR) cattle are the main dairy breed in Norway, bred according to a broad breeding goal including health and fertility since the 1970s. Genomic studies on NR cattle have relied on the public Hereford reference, thus increasing the risk of missing or misrepresenting NR breed-specific variation. Moreover, the Hereford reference is a pseudohaploid assembly, representing homologous chromosomes in a collapsed manner, which results in loss of haplotype-specific alleles and misrepresentation of complex variants. To develop more refined NR specific resources, we utilised long-read sequencing (PacBio HiFi + ONT) and trio-binning to construct six new haplotype-resolved assemblies representing NR genomes. These six NR2025 assemblies show high completeness (BUSCO: 95.82-98.11%) and contiguity (N50: 73.8-88.5 Mb) and are accurately phased (hamming error rate: 0.46-2.52%). Most autosomes have been assembled into the acrocentric centromere, and mapping of bovine satellite sequences reveal distinct organisational patterns of different satellite units across this highly repetitive region. The NR2025 assemblies provide a valuable resource for identification of novel variants and haplotypes in the NR population, which will enable more accurate association studies of genotype-to-phenotype relations and genomic predictions in NR cattle, ultimately enhancing the efficacy of selection for desirable traits.

Background: The analysis of single nucleotide polymorphism (SNP) genotypes which capture genome-wide polymorphic variation can be used to improve the accuracy of selection in breeding programs and reveal the genetic architecture of quantitative traits. Mid-density SNP genotyping arrays containing > 60 K SNP markers have been developed for numerous production species and have been used to enable enhanced breeding progress. Despite recent genomic advances, Atlantic halibut lacks mid-density genotyping tools, limiting the application of genomic selection and high-resolution association studies in this species. The evolving Atlantic halibut (Hippoglossus hippoglossus) industry is also interested in genetically informed selection of breeding candidates to facilitate genetic improvement. The present study reports the development of the first mid-density SNP-array for use in Atlantic halibut and describes its performance in samples from different breeding populations. Results: Variant discovery was performed using whole genome sequencing (WGS) data from 40 individuals and resulted in the detection of more than 8 million small genetic variants including 6,224,375 SNPs and 2,131,436 insertion-deletion variants (INDELS). An Affymetrix Axiom custom array containing 70,824 assays (including 697 for determining genetic gender) was created and used to genotype 1152 Atlantic halibut samples. Assays for 59,491 SNP loci (84% of the array total) performed well and generated reliable genotypes, with an average SNP call rate of 99.64% and genotype concordance of 99.0% with whole-genome sequencing data. Of the gender-specific loci, 133 SNP assays could be used to assign genetic gender. Conclusions: This article describes the first mid-density SNP genotyping array for Atlantic halibut. It contains nearly 60 K robust assays for genome-wide dispersed SNP loci, along with those suitable for determining genetic gender. The array is amenable for use as a platform for high-resolution genetics research into traits of evolutionary and economic importance and the results have practical relevance for genomic selection in Atlantic halibut.