Targeted sequencing for high-resolution evolutionary analyses following genome duplication in salmonid fish: Proof of concept for key components of the insulin-like growth factor axis

Abstract

High-throughput sequencing has revolutionised comparative and evolutionary genome biology. It has now become relatively commonplace to generate multiple genomes and/or transcriptomes to characterize the evolution of large taxonomic groups of interest. Nevertheless, such efforts may be unsuited to some research questions or remain beyond the scope of some research groups. Here we show that targeted high-throughput sequencing offers a viable alternative to study genome evolution across a vertebrate family of great scientific interest. Specifically, we exploited sequence capture and Illumina sequencing to characterize the evolution of key components from the insulin-like growth (IGF) signalling axis of salmonid fish at unprecedented phylogenetic resolution. The IGF axis represents a central governor of vertebrate growth and its core components were expanded by whole genome duplication in the salmonid ancestor ~95Ma. Using RNA baits synthesised to genes encoding the complete family of IGF binding proteins (IGFBP) and an IGF hormone (IGF2), we captured, sequenced and assembled orthologous and paralogous exons from species representing all ten salmonid genera. This approach generated 299 novel sequences, most as complete or near-complete protein-coding sequences. Phylogenetic analyses confirmed congruent evolutionary histories for all nineteen recognized salmonid IGFBP family members and identified novel salmonid-specific IGF2 paralogues. Moreover, we reconstructed the evolution of duplicated IGF axis paralogues across a replete salmonid phylogeny, revealing complex historic selection regimes - both ancestral to salmonids and lineage-restricted - that frequently involved asymmetric paralogue divergence under positive and/or relaxed purifying selection. Our findings add to an emerging literature highlighting diverse applications for targeted sequencing in comparative-evolutionary genomics. We also set out a viable approach to obtain large sets of nuclear genes for any member of the salmonid family, which should enable insights into the evolutionary role of whole genome duplication before additional nuclear genome sequences become available.

Lineage-specific rediploidization is a mechanism to explain time-lags between genome duplication and evolutionary diversification

“…To understand the extent and dynamics of lineage-specific rediploidization in salmonids, we used in-solution sequence capture [46] to generate a genome-wide ohnologue dataset spanning the salmonid phylogeny [32, 41]. Note, here we use the term ohnologue, but elsewhere ‘homeologue’ has been used to describe gene duplicates retained from the Ss4R WGD event [38].…”

“…Full methods were recently detailed elsewhere, including the source and selection of 16 study species [46]. While this past study was a small-scale investigation of a few genes [46], here we up-scaled the approach to 1293 unique capture probes (Additional file 5; Additional file 1: Text S3 provides details on probe design). 120mer oligomer baits were synthesised at fourfold tiling across the full probe set and a total of 1.5 Mbp of unique sequence data were produced in each capture library.…”

“…120mer oligomer baits were synthesised at fourfold tiling across the full probe set and a total of 1.5 Mbp of unique sequence data were produced in each capture library. The captures were performed on randomly fragmented gDNA libraries, meaning that the recovered data represent exons plus flanking genomic regions [46]. We recovered 21.7 million reads per species on average after filtering low-quality data (SD, 0.8 million reads; >99.1% paired-end data; Additional file 1: Table S3), which were assembled using SOAPdenovo2 [67] with a K-mer value of 91 and merging level of 3 (otherwise default parameters).…”

“…using BLASTn [68] and aligned with MAFFT v.7 using the default automatic strategy [70]. Northern pike was used as the outgroup to the Ss4R WGD in all analyses; this species was included in our target-enrichment study, but pike sequences were captured slightly less efficiently compared to salmonids [46]. Thus, we supplemented pike sequences using the latest genome assembly [47] (ASM72191v2; NCBI accession CF_000721915).…”

Growth hormone transgenesis disrupts immune function in muscle of coho salmon (Oncorhynchus kisutch) impacting cross-talk with growth systems

“…The action of IGFs is modulated in the extracellular environment by a family of IGF-binding proteins (IGFBPs) that influence the availability of these hormones to IGF-1R (Firth and Baxter, 2002). Many genes from the GH and IGF pathways have been expanded by genome duplication events in teleost evolutionary history (Ocampo Daza et al, 2011; Macqueen et al, 2013; Lappin et al, 2016; Alzaid et al, 2016a; Robertson et al, 2017), including a salmonid-specific event that occurred 88-103 Ma (Macqueen and Johnston, 2014).…”