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Phenotypic diversity and its genetic basis are central questions in biology, with domesticated animals offering valuable insights due to their rapid evolution the last 10,000 years. In chickens, fibromelanosis (FM) is a striking pigmentation phenotype characterized by hyperpigmentation. A previous study identified a complex structural variant involving both two large duplications (127.4 and 170.5 kb in size) and inversions associated with upregulated expression of the Endothelin 3 (EDN3) gene. However, the detailed organization of the structural arrangements have remained unclear. In this study, we conducted a comprehensive genomic survey of 517 FM chickens representing 44 different populations. Our results elucidate the complex arrangement of the duplications and inversions at the FM locus based on the large-scale genomic survey, population level genotyping, and linkage disequilibrium analysis, providing conclusive support for one specific configuration of the two large duplications, resolving a controversy that has been unresolved for more than a decade. Our results show that the birth of this complex structural variant must have involved an interchromosomal rearrangement creating fixed heterozygosity due to sequence differences between the two copies of the 127.4 kb duplication. This study shows how population genomics can be used to understand complex structural variations that underlie phenotypic variation.

Background

Snakes exhibit a broad variety of adaptive colors and color patterns, generated by the spatial arrangement of chromatophores, but little is known of the mechanisms responsible for these spectacular traits. Here, we investigate a mono-locus trait with two recessive alleles, motley and stripe, that both cause pattern aberrations in the corn snake.

Results

We use mapping-by-sequencing to identify the genomic interval where the causal mutations reside. With our differential gene expression analyses, we find that CLCN2 (Chloride Voltage-Gated Channel 2), a gene within the genomic interval, is significantly downregulated in Motley embryonic skin. Furthermore, we identify the stripe allele as the insertion of an LTR-retrotransposon in CLCN2, resulting in a disruptive mutation of the protein. We confirm the involvement of CLCN2 in color pattern formation by producing knock-out snakes that present a phenotype similar to Stripe. In humans and mice, disruption of CLCN2 results in leukoencephalopathy, as well as retinal and testes degeneration. Our single-cell transcriptomic analyses in snakes reveal that CLCN2 is indeed expressed in chromatophores during embryogenesis and in the adult brain, but the behavior and fertility of Motley and Stripe corn snakes are not impacted.

Conclusions

Our genomic, transcriptomic, and functional analyses identify a plasma membrane anion channel to be involved in color pattern development in snakes and show that an active LTR-retrotransposon might be a key driver of trait diversification in corn snakes.

The integument plays a critical role in functional adaptation, with macro-regional specification forming structures like beaks, combs, feathers, and scales, while micro-regional specification modifies skin appendage shapes. However, the molecular mechanisms remain largely unknown. Craniofacial integument displays dramatic diversity, exemplified by the Polish chicken (PC) with a homeotic transformation of comb-to-crest feathers, caused by a 195-base pair (bp) duplication in HoxC10 intron. Micro-C analyses show that HoxC-containing topologically associating domain (TAD) is normally closed in the scalp but open in the dorsal and tail regions, allowing multiple long-distance contacts. In the PC scalp, the TAD is open, resulting in high HoxC expression. CRISPR-Cas9 deletion of the 195-bp duplication reduces crest feather formation, and HoxC misexpression alters feather shapes. The 195-bp sequence is found only in Archelosauria (crocodilians and birds) and not in mammals. These findings suggest that higher-order regulation of the HoxC cluster modulates gene expression, driving the evolution of adaptive integumentary appendages in birds.

Understanding how marine organisms adapt to local environments is crucial for predicting how populations will respond to global climate change. The genomic basis, environmental factors and evolutionary processes involved in local adaptation are however not well understood. Here we use Atlantic herring, an abundant, migratory and widely distributed marine fish with substantial genomic resources, as a model organism to evaluate local adaptation. We examined genomic variation and its correlation with environmental variables across a broad environmental gradient, for 15 spawning aggregations in Atlantic Canada and the United States. We then compared our results with available genomic data of northeast Atlantic populations. We confirmed that population structure lies in a fraction of the genome including likely adaptive genetic variants of functional importance. We discovered 10 highly differentiated genomic regions distributed across four chromosomes. Nine regions show strong association with seasonal reproduction. One region, corresponding to a known inversion on chromosome 12, underlies a latitudinal pattern discriminating populations north and south of a biogeographic transition zone on the Scotian Shelf. Genome-environment associations indicate that winter seawater temperature best correlates with the latitudinal pattern of this inversion. The variation at two so-called 'islands of divergence' related to seasonal reproduction appear to be private to the northwest Atlantic. Populations in the northwest and northeast Atlantic share variation at four of these divergent regions, simultaneously displaying significant diversity in haplotype composition at another four regions, which includes an undescribed structural variant approximately 7.7 Mb long on chromosome 8. Our results suggest that the timing and geographic location of spawning and early development may be under diverse selective pressures related to allelic fitness across environments. Our study highlights the role of genomic architecture, ancestral haplotypes and selection in maintaining adaptive divergence in species with large population sizes and presumably high gene flow.

The Greying with age phenotype in horses involves loss of hair pigmentation whereas skin pigmentation is not reduced, and a predisposition to melanoma. The causal mutation was initially reported as a duplication of a 4.6kb intronic sequence in Syntaxin 17. The speed of greying varies considerably among Grey horses. Here we demonstrate the presence of two different Grey alleles, G2 carrying two tandem copies of the duplicated sequence and G3 carrying three. The latter is by far the most common allele, probably due to strong selection for the striking white phenotype. Our results reveal a remarkable dosage effect where the G3 allele is associated with fast greying and high incidence of melanoma whereas G2 is associated with slow greying and low incidence of melanoma. The copy number expansion transforms a weak enhancer to a strong melanocyte-specific enhancer that underlies hair greying (G2 and G3) and a drastically elevated risk of melanoma (G3 only). Our direct pedigree-based observation of the origin of a G2 allele from a G3 allele by copy number contraction demonstrates the dynamic evolution of this locus and provides the ultimate evidence for causality of the copy number variation of the 4.6kb intronic sequence.

The transcription factor and cell cycle regulator p53 is marked for degradation by the ubiquitin ligase MDM2. The interaction between these 2 proteins is mediated by a conserved binding motif in the disordered p53 transactivation domain (p53TAD) and the folded SWIB domain in MDM2. The conserved motif in p53TAD from zebrafish displays a 20-fold weaker interaction with MDM2, compared to the interaction in human and chicken. To investigate this apparent difference, we tracked the molecular evolution of the p53TAD/MDM2 interaction among ray-finned fishes (Actinopterygii), the largest vertebrate clade. Intriguingly, phylogenetic analyses, ancestral sequence reconstructions, and binding experiments showed that different loss-of-affinity changes in the canonical binding motif within p53TAD have occurred repeatedly and convergently in different fish lineages, resulting in relatively low extant affinities (KD = 0.5 to 5 mu M). However, for 11 different fish p53TAD/MDM2 interactions, nonconserved regions flanking the canonical motif increased the affinity 4- to 73-fold to be on par with the human interaction. Our findings suggest that compensating changes at conserved and nonconserved positions within the motif, as well as in flanking regions of low conservation, underlie a stabilizing selection of "functional affinity" in the p53TAD/MDM2 interaction. Such interplay complicates bioinformatic prediction of binding and calls for experimental validation. Motif-mediated protein-protein interactions involving short binding motifs and folded interaction domains are very common across multicellular life. It is likely that the evolution of affinity in motif-mediated interactions often involves an interplay between specific interactions made by conserved motif residues and nonspecific interactions by nonconserved disordered regions.

Background

Genome-wide comparisons of populations are widely used to explore the patterns of nucleotide diversity and sequence divergence to provide knowledge on how natural selection and genetic drift affect the genome. In this study we have compared whole-genome sequencing data from Atlantic and Pacific herring, two sister species that diverged about 2 million years ago, to explore the pattern of genetic differentiation between the two species.

Results

The genome comparison of the two species revealed high genome-wide differentiation but with islands of remarkably low genetic differentiation, as measured by an F_ST analysis. However, the low F_ST observed in these islands is not caused by low interspecies sequence divergence (d_xy) but rather by exceptionally high estimated intraspecies nucleotide diversity (π). These regions of low differentiation and elevated nucleotide diversity, termed high-diversity regions in this study, are not enriched for repeats but are highly enriched for immune-related genes. This enrichment includes genes from both the adaptive immune system, such as immunoglobulin, T-cell receptor and major histocompatibility complex genes, as well as a substantial number of genes with a role in the innate immune system, e.g. novel immune-type receptor, tripartite motif and tumor necrosis factor receptor genes. Analysis of long-read based assemblies from two Atlantic herring individuals revealed extensive copy number variation in these genomic regions, indicating that the elevated intraspecies nucleotide diversities were partially due to the cross-mapping of short reads.

Conclusions

This study demonstrates that copy number variation is a characteristic feature of immune trait loci in herring. Another important implication is that these loci are blind spots in classical genome-wide screens for genetic differentiation using short-read data, not only in herring, likely also in other species harboring qualitatively similar variation at immune trait loci. These loci stood out in this study because of the relatively high genome-wide baseline for F_ST values between Atlantic and Pacific herring.

Atlantic herring Clupea harengus feeding in the Norwegian Sea are assumed to consist of Norwegian spring spawners (NSSH), Icelandic summer spawners (ISSH) and North Sea autumn spawners (NSAH). Putative Norwegian autumn spawners (NASH), Faroese autumn (FASH) and spring (FSSH) spawners also feed in the area. However, until there is a method to discriminate between populations in mixed samples, fishery and survey data from the Norwegian Sea will be solely attributed to the predominating NSSH, ultimately causing biased stock assessments. Hence, we evaluated if a panel of 120 single nucleotide polymorphisms (SNPs) associated with spawning characteristics and salinity preferences would be an effective discrimination tool. The overall observed levels of genetic differentiation were high (F_ST = 0.57, p <0.001, 95% CI: 0.51-0.62). Spawners from stocks under current management (NSSH, NSAH and ISSH) were well separated, but the putative populations were not. Discriminant analysis of principal component as well as Structure runs confirmed the differentiation observed with F_ST. When the SNP panels were tested on commercial fishery samples of NSSH east of Iceland, up to 16% were assigned to ISSH. This implies that catch data are seriously biased and demonstrates the potential of SNP panels as a tool to solve the problem. However, work is needed to develop improved SNP panels that effectively separate the putative populations from the managed stocks. We recommend that such a tool should be established in regular sampling of fishery and surveys in the Norwegian Sea and accounted for in future stock assessments, advice and management.

The circumstances under which species diversify to genetically distinct lineages is a fundamental question in biology. Atlantic herring (Clupea harengus) is an extremely abundant zooplanktivorous species that is subdivided into multiple ecotypes that differ regarding spawning time and genetic adaption to local environmental conditions such as temperature, salinity, and light conditions. Here we show using whole genome analysis that multiple populations of piscivorous (fish-eating) herring have evolved sympatrically after the colonization of the brackish Baltic Sea within the last 8000 years postglaciation. The piscivorous ecotype grows faster, and is much larger and less abundant than the zooplanktivorous Baltic herring. Lesions of the gill rakers in the piscivorous ecotype indicated incomplete adaptation to a fish diet. This niche expansion of herring in the young Baltic Sea, with its paucity of piscivorous species, suggests that empty niche space is more important than geographic isolation for the evolution of biodiversity.

Sustainable fisheries management is important for the continued harvest of the world's marine resources, especially as they are increasingly challenged by a range of climatic and anthropogenic factors. One of the pillars of sustainable fisheries management is the accurate identification of the biological units, i.e., populations. Here, we developed and implemented a genetic baseline for Atlantic herring harvested in the Norwegian offshore fisheries to investigate the validity of the current management boundaries. This was achieved by genotyping > 15,000 herring from the northern European seas, including samples of all the known populations in the region, with a panel of population-informative SNPs mined from existing genomic resources. The final genetic baseline consisted of similar to 1000 herring from 12 genetically distinct populations. We thereafter used the baseline to investigate mixed catches from the North and Norwegian Seas, revealing that each management area consisted of multiple populations, as previously suspected. However, substantial numbers (up to 50% or more within a sample) of herring were found outside of their expected management areas, e.g., North Sea autumn-spawning herring north of 62 degrees N (average = 19.2%), Norwegian spring-spawning herring south of 62 degrees N (average = 13.5%), and western Baltic spring-spawning herring outside their assumed distribution area in the North Sea (average = 20.0%). Based upon these extensive observations, we conclude that the assessment and management areas currently in place for herring in this region need adjustments to reflect the populations present. Furthermore, we suggest that for migratory species, such as herring, a paradigm shift from using static geographic stock boundaries towards spatial dynamic boundaries is needed to meet the requirements of future sustainable management regimes.