mRNA Extraction from Gill Tissue for RNA-sequencing

Adaptation is thought to proceed in part through spatial and temporal changes in gene expression. Fish species such as the threespine stickleback are powerful vertebrate models to study the genetic architecture of adaptive changes in gene expression since divergent adaptation to different environments is common, they are abundant and easy to study in the wild and lab, and have well-established genetic and genomic resources. Fish gills, due to their respiratory and osmoregulatory roles, show many physiological adaptations to local water chemistry, including differences in gene expression. However, obtaining high-quality RNA using popular column-based extraction methods can be challenging from small tissue samples high in cartilage and bone such as fish gills. Here, we describe a bead-based mRNA extraction and transcriptome RNA-seq protocol that does not use purification columns. The protocol can be readily scaled according to sample size for the purposes of diverse gene expression experiments using animal or plant tissue.Peer reviewe

Predominance of cis-regulatory changes in parallel expression divergence of sticklebacks

Regulation of gene expression is thought to play a major role in adaptation but there are conflicting predictions for the relative importance of cis- and trans-regulatory mechanisms in the early stages adaptive divergence. Parallel evolution of marine and freshwater threespine stickleback fish provides an excellent opportunity to dissect whether the same molecular mechanisms underlie repeated adaptive divergence in gene expression. Using RNA-seq of four marine-freshwater ecotype pairs from Scotland and Canada, we first identified genes with parallel divergence in expression and show that these are found near previously reported adaptive loci and show a molecular signature of selection centered around the transcription start site. With allele-specific expression assays in F1 hybrids we next show that expression divergence is predominantly driven by cis-regulatory control in all four river systems, a pattern that is enriched in parallel divergently expressed genes. In particular, for genes whose expression is up-regulated in parallel among freshwater fish the quantitative degree of cis- and trans-regulation is also highly correlated, suggesting a shared genetic basis across populations. This stands in contrast to genes up-regulated in parallel in marine fish, whose degree of cis- and trans-regulation is less correlated and predictable. This observed asymmetry in parallelism in how genes are up-regulated in marine and freshwater fish can be explained by differences in the evolutionary contexts of the diverging ecotypes. Finally, we show that cis-regulation is predominantly additive and shows greater robustness to different in genetic backgrounds and environmental conditions. We argue that these features make cis-regulation well-poised for rapid adaptive divergence of gene expression under conditions of on-going gene flow. Combined our study highlights how natural selection on dispersed cis-regulatory elements can shape the adaptive landscape of the genome.Regulation of gene expression is thought to play a major role in adaptation but the relative importance of cis- and trans- regulatory mechanisms in the early stages of adaptive divergence is unclear. Using RNAseq of threespine stickleback fish gill tissue from four independent marine-freshwater ecotype pairs and their F1 hybrids, we show that cis-acting (allele-specific) regulation consistently predominates gene expression divergence. Genes showing parallel marine-freshwater expression divergence are found near to adaptive genomic regions, show signatures of natural selection around their transcription start sites and are enriched for cis-regulatory control. For genes with parallel increased expression among freshwater fish, the quantitative degree of cis- and trans-regulation is also highly correlated across populations, suggesting a shared genetic basis. Compared to other forms of regulation, cis-regulation tends to show greater additivity and stability across different genetic and environmental contexts, making it a fertile substrate for the early stages of adaptive evolution.Peer reviewe

Genetic Drift Dominates Genome-Wide Regulatory Evolution Following an Ancient Whole-Genome Duplication in Atlantic Salmon

Whole-genome duplications (WGD) have been considered as springboards that potentiate lineage diversification through increasing functional redundancy. Divergence in gene regulatory elements is a central mechanism for evolutionary diversification, yet the patterns and processes governing regulatory divergence following events that lead to massive functional redundancy, such as WGD, remain largely unknown. We studied the patterns of divergence and strength of natural selection on regulatory elements in the Atlantic salmon (Salmo salar) genome, which has undergone WGD 100–80 Ma. Using ChIPmentation, we first show that H3K27ac, a histone modification typical to enhancers and promoters, is associated with genic regions, tissue-specific transcription factor binding motifs, and with gene transcription levels in immature testes. Divergence in transcription between duplicated genes from WGD (ohnologs) correlated with difference in the number of proximal regulatory elements, but not with promoter elements, suggesting that functional divergence between ohnologs after WGD is mainly driven by enhancers. By comparing H3K27ac regions between duplicated genome blocks, we further show that a longer polyploid state post-WGD has constrained regulatory divergence. Patterns of genetic diversity across natural populations inferred from resequencing indicate that recent evolutionary pressures on H3K27ac regions are dominated by largely neutral evolution. In sum, our results suggest that post-WGD functional redundancy in regulatory elements continues to have an impact on the evolution of the salmon genome, promoting largely neutral evolution of regulatory elements despite their association with transcription levels. These results highlight a case where genome-wide regulatory evolution following an ancient WGD is dominated by genetic drift.Peer reviewe

The Atlantic salmon whole blood transcriptome and how it relates to major locus maturation genotypes and other tissues

The Atlantic salmon (Salmo salar) is important to many ecosystems and local economies and has therefore become the focus of a broad range of research questions that have benefited from the availability of high-quality genomic resources. Albeit gene expression studies have been extensive for this species, the transcriptome information for Atlantic salmon whole blood has been lacking. A transcriptome of Atlantic salmon blood would be a valuable resource for future studies, especially those wishing to take non-lethal samples. Here, we report a whole blood transcriptome for Atlantic salmon constructed from twelve 8-month old salmon parr using RNA-seq. We identify transcriptomic proxies for the genotype at the major maturation timing locus vestigial-like 3 (vgll3). Differentially expressed genes between the early and late maturing genotypes showed overrepresented Gene Ontology (GO) terms with the strongest result linked to 13 ribosomal subunit genes. To assess how the whole blood gene expression profile relates to other tissues, we compare the blood transcriptome to the reference transcriptome of fourteen other tissue types using both a common PCA method and a novel method. The novel method compares transcriptomes when gene expression is visualised as a layer using thin-plate spline smoothers. Both methods found similar patterns with the blood transcriptome being quite unique compared to the transcription profiles of other tissues.Peer reviewe

Utilization of Tissue Ploidy Level Variation in de Novo Transcriptome Assembly of Pinus sylvestris

Compared to angiosperms, gymnosperms lag behind in the availability of assembled and annotated genomes. Most genomic analyses in gymnosperms, especially conifer tree species, rely on the use of de novo assembled transcriptomes. However, the level of allelic redundancy and transcript fragmentation in these assembled transcriptomes, and their effect on downstream applications have not been fully investigated. Here, we assessed three assembly strategies for short-reads data, including the utility of haploid megagametophyte tissue during de novo assembly as single-allele guides, for six individuals and five different tissues in Pinus sylvestris. We then contrasted haploid and diploid tissue genotype calls obtained from the assembled transcriptomes to evaluate the extent of paralog mapping. The use of the haploid tissue during assembly increased its completeness without reducing the number of assembled transcripts. Our results suggest that current strategies that rely on available genomic resources as guidance to minimize allelic redundancy are less effective than the application of strategies that cluster redundant assembled transcripts. The strategy yielding the lowest levels of allelic redundancy among the assembled transcriptomes assessed here was the generation of SuperTranscripts with Lace followed by CD-HIT clustering. However, we still observed some levels of heterozygosity (multiple gene fragments per transcript reflecting allelic redundancy) in this assembled transcriptome on the haploid tissue, indicating that further filtering is required before using these assemblies for downstream applications. We discuss the influence of allelic redundancy when these reference transcriptomes are used to select regions for probe design of exome capture baits and for estimation of population genetic diversity.Peer reviewe

Cis-regulatory differences in isoform expression associate with life history strategy variation in Atlantic salmon

A major goal in biology is to understand how evolution shapes variation in individual life histories. Genome-wide association studies have been successful in uncovering genome regions linked with traits underlying life history variation in a range of species. However, lack of functional studies of the discovered genotype-phenotype associations severely restrains our understanding how alternative life history traits evolved and are mediated at the molecular level. Here, we report acis-regulatory mechanism whereby expression of alternative isoforms of the transcription co-factorvestigial-like 3(vgll3) associate with variation in a key life history trait, age at maturity, in Atlantic salmon (Salmo salar). Using a common-garden experiment, we first show thatvgll3genotype associates with puberty timing in one-year-old salmon males. By way of temporal sampling ofvgll3expression in ten tissues across the first year of salmon development, we identify a pubertal transition invgll3expression where maturation coincided with a 66% reduction in testicularvgll3expression. Thelatematuration allele was not only associated with a tendency to delay puberty, but also with expression of a rare transcript isoform ofvgll3pre-puberty. By comparing absolutevgll3mRNA copies in heterozygotes we show that the expression difference between theearlyandlatematurity alleles is largelycis-regulatory. We propose a model whereby expression of a rare isoform from thelateallele shifts the liability of its carriers towards delaying puberty. These results exemplify the potential importance of regulatory differences as a mechanism for the evolution of life history traits. Author summary Alternative life history strategies are an important source of diversity within populations and promote the maintenance of adaptive capacity and population resilience. However, in many cases the molecular basis of different life history strategies remains elusive. Age at maturity is a key adaptive life history trait in Atlantic salmon and has a relatively simple genetic basis. Using salmon age at maturity as a model, we report a mechanism whereby different transcript isoforms of the key age at maturity gene,vestigial-like 3(vgll3), associate with variation in the timing of male puberty. Our results show how gene regulatory differences in conjunction with variation in gene transcript structure can encode for complex alternative life histories.Peer reviewe

Maturation in Atlantic salmon (Salmo salar, Salmonidae) : a synthesis of ecological, genetic, and molecular processes

Over the past decades, Atlantic salmon (Salmo salar, Salmonidae) has emerged as a model system for sexual maturation research, owing to the high diversity of life history strategies, knowledge of trait genetic architecture, and their high economic value. The aim of this synthesis is to summarize the current state of knowledge concerning maturation in Atlantic salmon, outline knowledge gaps, and provide a roadmap for future work. We summarize the current state of knowledge: 1) maturation in Atlantic salmon takes place over the entire life cycle, starting as early as embryo development, 2) variation in the timing of maturation promotes diversity in life history strategies, 3) ecological and genetic factors influence maturation, 4) maturation processes are sex-specific and may have fitness consequences for each sex, 5) genomic studies have identified large-effect loci that influence maturation, 6) the brain-pituitary-gonadal axis regulates molecular and physiological processes of maturation, 7) maturation is a key component of fisheries, aquaculture, conservation, and management, and 8) climate change, fishing pressure, and other anthropogenic stressors likely have major effects on salmon maturation. In the future, maturation research should focus on a broader diversity of life history stages, including early embryonic development, the marine phase and return migration. We recommend studies combining ecological and genetic approaches will help disentangle the relative contributions of effects in different life history stages to maturation. Functional validation of large-effect loci should reveal how these genes influence maturation. Finally, continued research in maturation will improve our predictions concerning how salmon may adapt to fisheries, climate change, and other future challenges.Peer reviewe

Genetics of gene expression in conifers

Décrire les bases génétiques de la variation phénotypique est un but important en biologie; c’est aussi nécessaire pour comprendre l’évolution et développer des outils utilisables en amélioration et conservation. L’expression des gènes, mesurée par le niveau de transcrits produits, reflète l’activité des gènes et la variation de cette expression influence les phénotypes moléculaire et physiologiques en aval. Cette thèse présente deux expériences visant à développer une approche complémentaire aux méthodes traditionnelles souvent inapplicables chez les conifères afin d'étudier les bases génétiques de la variation d’expression génique chez l’épinette blanche (Picea glauca, Moench. Voss). Analyses des tissus haploïde et diploïde de semences à l’aide de biopuces d'expression et de RNA-seq ont permis de décrire la variation d’expression selon la complexité des bases génétiques, la fonction biologique des gènes impliqués, la liaison génétique entre les effets et les gènes influencés, la spécificité des effets génétiques aux allèles, et l’additivité des effets génétiques chez des individus hétérozygotes. Les effets ayant des bases génétiques relativement simples étaient majoritairement uniques à chaque individu et surreprésentés au sein des gènes impliqués dans la réponse au stress et les gènes dupliqués, suggérant un rôle adaptif. Les effets liés aux variations génétiques situées au sein du gène peuvent être particulièrement importants pour l’adaptation en raison de leur plus grande additivité et spécificité allélique. Une minorité des effets locaux étaient non-spécifiques aux allèles, en accord avec une action de facteurs de régulations indépendants mais étroitement liés au gène ou une auto-régulation de la transcription par le gène lui-même. Une grande partie de la variation d'expression locale montrait des signes de compensation entre des effets spécifiques et non-spécifiques aux allèles, possiblement en raison d'une sélection qui favoriserait un lien étroit entre les effets agissant sur le même gène. Les effets non-liés, non-spécifiques aux allèles, étaient transmis de façon cohérente avec la théorie physiologique de Wright sur la dominance, suggérant qu'ils modulent l'expression en interaction avec d'autres facteurs. L’approche développée dans cette thèse peut potentiellement être appliquée à la large gamme des conifères, avec le possibilité d’étudier le rôle de l'expression dans la divergence entre les populations et les espèces.Dissecting the genetic bases of phenotypic variation is a major goal in biology and underlies basic understanding of evolution as well as development of practical applications in breeding and conservation. Gene expression (transcript accumulation) reflects gene activity and variation therein translates to variation in other molecular and physiological phenotypes. The goal of my thesis was to establish a novel system for the genetic dissection of expression variation in white spruce (Picea glauca, Moench. Voss) because conventional approaches are mostly unfeasible in conifers. The approach that was used in developing this system is based on analyzing gene expression in the haploid and diploid meiotic seed tissues. Expression analysis using both microarray and RNA-seq techniques revealed a complex and diverse genetic basis for expression variation, which was dissected according to the complexity of genetic bases, focal gene function, effect linkage, effect allele-specificity and additivity of expression variation in heterozygous individuals. Expression variation with simple genetic bases was largely unique to each of two studied individuals and was biased towards genes involved in stress-response and duplicated genes, indicating that it may have adaptive value. Genetic variation in linkage with the expressed gene may be particularly important in adaptive evolution because of their additive effects, associated with their allele-specificity. A minority of local effects was non-specific to alleles, consistent with the action of closely linked but independent regulatory factors or self-regulation of transcription. Much of local expression variation showed evidence of compensation by allele-specific and non-specific effects, suggesting that selection has favored close linkage between certain effets acting on the same gene. Unlinked effects, always non-specific to alleles, exhibited inheritance consistent with Wright’s physiological theory of dominance suggesting that they influence expression in interaction with other partners. Furthermore, widespread dominance/recessivity in expression variation may explain the observation of highly pleiotropic effects influencing key cellular processes and suggests that recessive expression variation may contribute to inbreeding depression. The approach developed in this thesis can potentially be applied to other conifers because of their shared reproductive biology, and can be used to address the role of expression variation in the divergence of populations and species