Copy number variation and elevated genetic diversity at immune trait loci in Atlantic and Pacific herring.

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 FST analysis. However, the low FST observed in these islands is not caused by low interspecies sequence divergence (dxy) 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 FST values between Atlantic and Pacific herring.

Ecological adaptation in Atlantic herring is associated with large shifts in allele frequencies at hundreds of loci.

Atlantic herring is widespread in North Atlantic and adjacent waters and is one of the most abundant vertebrates on earth. This species is well suited to explore genetic adaptation due to minute genetic differentiation at selectively neutral loci. Here, we report hundreds of loci underlying ecological adaptation to different geographic areas and spawning conditions. Four of these represent megabase inversions confirmed by long read sequencing. The genetic architecture underlying ecological adaptation in herring deviates from expectation under a classical infinitesimal model for complex traits because of large shifts in allele frequencies at hundreds of loci under selection.

Unraveling the evolutionary origin of ELR motif using fish CXC chemokine CXCL8.

Chemokines are chemotactic proteins involved in host defense through the migration of immune-regulatory cells to the site of infection. Interleukin-8 (CXCL8/IL8) is the most studied "ELR-CXC chemokine/neutrophil activating chemokine (NAC) that regulate neutrophil trafficking during infections and inflammation by binding to its cognate G-protein coupled receptors CXCR1/CXCR2. The "ELR" motif of NAC chemokines is essential for the CXCR1/CXCR2 receptor activation. In order to understand the evolutionary origin of "ELR" motif in the CXC chemokines, a thorough evolutionary study of CXCL8 gene from various fishes and primates was performed. Phylogenetic analysis revealed that the CXCL8 gene can be classified into four distinct lineages (CXCL8-L1a, CXCL8-L1b, CXCL8-L2, and CXCL8-L3), where CXCL8-L1a is the fastest evolving lineage and CXCL8-L3 is the slowest. Selection analysis suggested that The "ELR/DLR" motif containing branches (gadoid and coelacanth) are positively selected. The probable evolutionary trend of "ELR" motif suggested that this motif in ancestor CXCL8 is evolved from the GGR of Lamprey (Agnatha), followed by duplication giving rise to two main motifs in CXCL8 "NXH" in L3 lineage and "ELR/DLR" in L1a/L1b lineages. Although, structural analysis suggested that the overall topology of the CXCL8 proteins is similar, differences do exist at the individual structural elements among the members of different lineages. Functional distance analysis suggested that the CXCL8-L3 lineage is more distant compared to the CXCL8-L1a and L1b lineages from the inferred ancestor. Functional divergence analysis between different lineages suggested that most of the selected residues are important for receptor or glycosaminoglycan binding. Such a functional diversification can be attributed to the novel set of functions adopted by CXCL8 in various species.

Molecular cloning and biophysical characterization of CXCL3 chemokine.

CXCL3 is a neutrophil activating chemokine that belongs to GRO subfamily of CXC chemokines. GRO chemokine family comprises of three chemokines GRO α (CXCL1), GROß (CXCL2), and GRO γ (CXCL3), which arose as a result of gene duplication events during the course of chemokine evolution. Although primary sequences of GRO chemokines are highly similar, they performs several protein specific functions in addition to their common property of neutrophil trafficking. However, the molecular basis for their differential functions has not well understood. Although structural details are available for CXCL1 and CXCL2, no such information regarding CXCL3 is available till date. In the present study, we have successfully cloned, expressed, and purified the recombinant CXCL3. Around 15mg/L of pure recombinant CXCL3 protein was obtained. Further, we investigated its functional divergence and biophysical characteristics such as oligomerization, thermal stability and heparin binding etc., and compared all these features with its closest paralog CXCL2. Our studies revealed that, although overall structural and oligomerization features of CXCL3 and CXCL2 are similar, prominent differences were observed in their surface characteristics, thus implicating for a functional divergence.

Mechanistic insights into molecular evolution of species-specific differential glycosaminoglycan binding surfaces in growth-related oncogene chemokines.

Chemokines are chemotactic cytokines involved in leucocyte trafficking to infected tissue. Growth-related oncogene (GRO) chemokines namely CXCL1, CXCL2 and CXCL3 are neutrophil activating chemokines sharing a conserved three-dimensional structure, but encompassing functional diversity due to gene duplication and evolutionary events. However, the evolutionary mechanisms including selection pressures involved in diversification of GRO genes have not yet been characterized. Here, we performed comprehensive evolutionary analysis of GRO genes among different mammalian species. Phylogenetic analysis illustrated a species-specific evolution pattern. Selection analysis evidenced that these genes have undergone concerted evolution. Seventeen positively selected sites were obtained, although the majority of the protein is under purifying selection. Interestingly, these positively selected sites are more concentrated on the C-terminal/glycosaminoglycan (GAG) binding and dimerization segment compared to receptor binding domain. Substitution rate analysis confirmed the C-terminal domain of GRO genes as the highest substituted segment. Further, structural analysis established that the nucleotide alterations in the GAG binding domain are the source of surface charge modulation, thus generating the differential GAG binding surfaces and multiple binding sites as per evolutionary pressure, although the helical surface is primordial for GAG binding. Indeed, such variable electrostatic surfaces are crucial to regulate chemokine gradient formation during a host's defence against pathogens and also explain the significance of chemokine promiscuity.