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Abstract: Large genomic studies are becoming increasingly common with advances in sequencing technology, and our ability to understand how genomic variation influences phenotypic variation between individuals has never been greater. The exploration of such relationships first requires the identification of associations between molecular markers and phenotypes. Here, we explore the use of Random Forest (RF), a powerful machine‐learning algorithm, in genomic studies to discern loci underlying both discrete and quantitative traits, particularly when studying wild or nonmodel organisms. RF is becoming increasingly used in ecological and population genetics because, unlike traditional methods, it can efficiently analyse thousands of loci simultaneously and account for nonadditive interactions. However, understanding both the power and limitations of Random Forest is important for its proper implementation and the interpretation of results. We therefore provide a practical introduction to the algorithm and its use for identifying associations between molecular markers and phenotypes, discussing such topics as data limitations, algorithm initiation and optimization, as well as interpretation. We also provide short R tutorials as examples, with the aim of providing a guide to the implementation of the algorithm. Topics discussed here are intended to serve as an entry point for molecular ecologists interested in employing Random Forest to identify trait associations in genomic data sets. [ABSTRACT FROM AUTHOR]

Background: Increased availability of genome assemblies for non-model organisms has resulted in invaluable biological and genomic insight into numerous vertebrates, including teleosts. Sequencing of the Atlantic cod (Gadus morhua) genome and the genomes of many of its relatives (Gadiformes) demonstrated a shared loss of the major histocompatibility complex (MHC) II genes 100 million years ago. An improved version of the Atlantic cod genome assembly shows an extreme density of tandem repeats compared to other vertebrate genome assemblies. Highly contiguous assemblies are therefore needed to further investigate the unusual immune system of the Gadiformes, and whether the high density of tandem repeats found in Atlantic cod is a shared trait in this group. Results: Here, we have sequenced and assembled the genome of haddock (Melanogrammus aeglefinus) – a relative of Atlantic cod – using a combination of PacBio and Illumina reads. Comparative analyses reveal that the haddock genome contains an even higher density of tandem repeats outside and within protein coding sequences than Atlantic cod. Further, both species show an elevated number of tandem repeats in genes mainly involved in signal transduction compared to other teleosts. A characterization of the immune gene repertoire demonstrates a substantial expansion of MCHI in Atlantic cod compared to haddock. In contrast, the Toll-like receptors show a similar pattern of gene losses and expansions. For the NOD-like receptors (NLRs), another gene family associated with the innate immune system, we find a large expansion common to all teleosts, with possible lineage-specific expansions in zebrafish, stickleback and the codfishes. Conclusions: The generation of a highly contiguous genome assembly of haddock revealed that the high density of short tandem repeats as well as expanded immune gene families is not unique to Atlantic cod – but possibly a feature common to all, or most, codfishes. A shared expansion of NLR genes in teleosts suggests that the NLRs have a more substantial role in the innate immunity of teleosts than other vertebrates. Moreover, we find that high copy number genes combined with variable genome assembly qualities may impede complete characterization of these genes, i.e. the number of NLRs in different teleost species might be underestimates. [ABSTRACT FROM AUTHOR]

Anadromous Chinook salmon populations vary in the period of river entry at the initiation of adult freshwater migration, facilitating optimal arrival at natal spawning. Run timing is a polygenic trait that shows evidence of rapid parallel evolution in some lineages, signifying a key role for this phenotype in the ecological divergence between populations. Studying the genetic basis of local adaptation in quantitative traits is often impractical in wild populations. Therefore, we used a novel approach, Random Forest, to detect markers linked to run timing across 14 populations from contrasting environments in the Columbia River and Puget Sound, USA. The approach permits detection of loci of small effect on the phenotype. Divergence between populations at these loci was then examined using both principle component analysis and FST outlier analyses, to determine whether shared genetic changes resulted in similar phenotypes across different lineages. Sequencing of 9107 RAD markers in 414 individuals identified 33 predictor loci explaining 79.2% of trait variance. Discriminant analysis of principal components of the predictors revealed both shared and unique evolutionary pathways in the trait across different lineages, characterized by minor allele frequency changes. However, genome mapping of predictor loci also identified positional overlap with two genomic outlier regions, consistent with selection on loci of large effect. Therefore, the results suggest selective sweeps on few loci and minor changes in loci that were detected by this study. Use of a polygenic framework has provided initial insight into how divergence in a trait has occurred in the wild. [ABSTRACT FROM AUTHOR]

Studying the actions of selection provides insight into adaptation, population divergence and gene function. Next-generation sequencing produces large amounts of partial sequences, potentially facilitating efforts to detect signatures of selection based on comparisons between synonymous ( d) and nonsynonymous ( d) substitutions, and single nucleotide polymorphism assays placed in selected genes would improve the ability to study adaptation in population surveys. However, sequences generated by these technologies are typically short. In nonmodel organisms that are a focus of evolutionary studies, the lack of a reference genome that facilitates the assembly of short sequences has limited surveys of positive selection in large numbers of genes. Here, we describe a series of steps to facilitate these surveys. We provide scripts to assist data analysis, and describe the use of commonly available programs. We demonstrate these approaches in six salmon species, which have partially duplicated genomes. We recommend using multiway to optimize the number of alignments between partial coding sequences. Reading frames should be manually detected after alignment with sequences in Genbank using the program. We encourage the use of a phylogenetic approach to separate orthologs from paralogs in duplicated genomes. Simple simulations on a gene known to have undergone selection in salmon species, transferrin, showed that the ability to detect selection in short sequences (<600 bp) depended on the proportion of codons under selection (1-2%) within that sequence. This relationship was less relevant in longer sequences. In this exploratory study, we detected 11 genes showing evidence of positive selection. [ABSTRACT FROM AUTHOR]

Abstract: A novel application of genomewide association analyses is to use trait‐associated loci to monitor the effects of conservation strategies on potentially adaptive genetic variation. Comparisons of fitness between captive‐ and wild‐origin individuals, for example, do not reveal how captive rearing affects genetic variation underlying fitness traits or which traits are most susceptible to domestication selection. Here, we used data collected across four generations to identify loci associated with six traits in adult Chinook salmon (Oncorhynchus tshawytscha) and then determined how two alternative management approaches for captive rearing affected variation at these loci. Loci associated with date of return to freshwater spawning grounds (return timing), length and weight at return, age at maturity, spawn timing, and daily growth coefficient were identified using 9108 restriction site‐associated markers and random forest, an approach suitable for polygenic traits. Mapping of trait‐associated loci, gene annotations, and integration of results across multiple studies revealed candidate regions involved in several fitness‐related traits. Genotypes at trait‐associated loci were then compared between two hatchery populations that were derived from the same source but are now managed as separate lines, one integrated with and one segregated from the wild population. While no broad‐scale change was detected across four generations, there were numerous regions where trait‐associated loci overlapped with signatures of adaptive divergence previously identified in the two lines. Many regions, primarily with loci linked to return and spawn timing, were either unique to or more divergent in the segregated line, suggesting that these traits may be responding to domestication selection. This study is one of the first to utilize genomic approaches to demonstrate the effectiveness of a conservation strategy, managed gene flow, on trait‐associated—and potentially adaptive—loci. The results will promote the development of trait‐specific tools to better monitor genetic change in captive and wild populations. [ABSTRACT FROM AUTHOR]

Captive breeding has the potential to rebuild depressed populations. However, associated genetic changes may decrease restoration success and negatively affect the adaptive potential of the entire population. Thus, approaches that minimize genetic risks should be tested in a comparative framework over multiple generations. Genetic diversity in two captive-reared lines of a species of conservation interest, Chinook salmon ( Oncorhynchus tshawytscha), was surveyed across three generations using genome-wide approaches. Genetic divergence from the source population was minimal in an integrated line, which implemented managed gene flow by using only naturally-born adults as captive broodstock, but significant in a segregated line, which bred only captive-origin individuals. Estimates of effective number of breeders revealed that the rapid divergence observed in the latter was largely attributable to genetic drift. Three independent tests for signatures of adaptive divergence also identified temporal change within the segregated line, possibly indicating domestication selection. The results empirically demonstrate that using managed gene flow for propagating a captive-reared population reduces genetic divergence over the short term compared to one that relies solely on captive-origin parents. These findings complement existing studies of captive breeding, which typically focus on a single management strategy and examine the fitness of one or two generations. [ABSTRACT FROM AUTHOR]