A genetic linkage map for the salmon louse (Lepeophtheirus salmonis): evidence for high male:female and inter-familial recombination rate differences.

A salmon louse (Lepeophtheirus salmonis salmonis) genetic linkage map was constructed to serve as a genomic resource for future investigations into the biology of this important marine parasitic copepod species, and to provide insights into the inheritance patterns of genetic markers in this species. SNP genotyping of 8 families confirmed the presence of 15 linkage groups based upon the assignment of 93,773 markers. Progeny sample size weight adjusted map sizes in males (with the exception of SL12 and SL15) ranged in size from 96.50 cM (SL11) to 134.61 cM (SL06), and total combined map steps or bins ranged from 143 (SL09) to 203 (SL13). The SL12 male map was the smallest linkage group with a weight-averaged size of 3.05 cM with 6 recombination bins. Male:female specific recombination rate differences are 10.49:1 and represent one of the largest reported sex-specific differences for any animal species. Recombination ratio differences (M:F) ranged from 1.0 (SL12) to 29:1 (SL15). The number of markers exhibiting normal Mendelian segregation within the sex linkage group SL15 was extremely low (N = 80) in comparison to other linkage groups genotyped [range: 1459 (SL12)-10206 markers (SL05)]. Re-evaluation of Mendelian inheritance patterns of markers unassigned to any mapping parent according to hemizygous segregation patterns (models presented) identified matches for many of these markers to hemizygous patterns. The greatest proportion of these markers assigned to SL15 (N increased to 574). Inclusion of the hemizygous markers revised SL15 sex-specific recombination rate differences to 28:1. Recombination hot- and coldspots were identified across all linkage groups with all linkage groups possessing multiple peaks. Nine of 13 linkage groups evaluated possessed adjacent domains with hot-coldspot transitional zones. The most common pattern was for one end of the linkage to show elevated recombination in addition to internal regions. For SL01 and SL06, however, a terminal region with high recombination was not evident while a central domain possessing extremely high-recombination levels was present. High levels of recombination were weakly coupled to higher levels of SNP variation within domains, but this association was very strong for the central domains of SL01 and SL06. From the pooled paternal half-sib lots (several virgin females placed with 1 male), only 1 or two surviving family lots were obtained. Surviving families possessed parents where both the male and female possessed either inherently low or high recombination rates. This study provides insight into the organization of the sea louse genome, and describes large differences in recombination rate that exist among individuals of the same sex, and between the sexes. These differences in recombination rate may be coupled to the capabilities of this species to adapt to environmental and pharmaceutical treatments, given that family survivorship appears to be enhanced when parents have similar recombination levels.

Transcriptome profiling in fast versus slow-growing rainbow trout across seasonal gradients.

BACKGROUND: Circannual rhythms in vertebrates can influence a wide variety of physiological processes. Some notable examples include annual reproductive cycles and for poikilotherms, seasonal changes modulating growth. Increasing water temperature elevates growth rates in fishes, but increases in photoperiod regime can have similar influences even at constant temperature. Therefore, in order to understand the dynamics of growth in fish it is important to consider the background influence of photoperiod regime on gene expression differences. This study examined the influence of a declining photoperiod regime (winter solstice) compared to an increasing photoperiod regime (spring equinox) on white muscle transcriptome profiles in fast and slow-growing rainbow trout from a commercial aquaculture strain. RESULTS: Slow-growing fish could be characterized as possessing transcriptome profiles that conform in many respects to an endurance training regime in humans. They have elevated mitochondrial and cytosolic creatine kinase expression levels and appear to suppress mTOR-signaling as evidenced by elevated TSC2 expression, and they also have elevated p53 levels. Large fish display a physiological repertoire that may be consistent with strength/resistance physiology having elevated cytoskeletal gene component expression and glycogen metabolism cycling along with higher PI3K levels. In many respects small vs. large fish match eccentric vs. concentric muscle expression patterns, respectively. Lipid metabolic genes are also more elevated in larger fish, the most notable being the G0S2 switch gene. M and Z-line sarcomere remodelling appears to be more prevalent in large fish. Twenty-three out of 26 gene families with previously reported significant SNP-based growth differences were detected as having significant expression differences. CONCLUSIONS: Larger fish display a broader array of genes showing higher expression, and their profiles are more similar to those observed in December lot fish (i.e., an accelerated growth period). Conversely, small fish display gene profiles more similar to seasonal growth decline phases (i.e., September lot fish). Overall, seasonal timing was coupled to greater differences in gene expression compared to differences associated with fish size.

Transcriptomics of salinity tolerance capacity in Arctic charr (Salvelinus alpinus): a comparison of gene expression profiles between divergent QTL genotypes.

Osmoregulatory capabilities have played an important role in the evolution, dispersal, and diversification of vertebrates. To better understand the genetic architecture of hypo-osmoregulation in fishes and to determine which genes and biological processes affect intraspecific variation in salinity tolerance, we used mRNA sequence libraries from Arctic charr gill tissue to compare gene expression profiles in fish exhibiting divergent salinity tolerance quantitative trait locus (QTL) genotypes. We compared differentially expressed genes with QTL positions to gain insight about the nature of the underlying polymorphisms and examined gene expression within the context of genome organization to gain insight about the evolution of hypo-osmoregulation in fishes. mRNA sequencing of 18 gill tissue libraries produced 417 million reads, and the final reduced de novo transcriptome assembly consisted of 92,543 contigs. Families contained a similar number of differentially expressed contigs between high and low salinity tolerance capacity groups, and log2 expression ratios ranged from 10.4 to -8.6. We found that intraspecific variation in salinity tolerance capacity correlated with differential expression of immune response genes. Some differentially expressed genes formed clusters along linkage groups. Most clusters comprised gene pairs, though clusters of three, four, and eight genes were also observed. We postulated that conserved synteny of gene clusters on multiple ancestral and teleost chromosomes may have been preserved via purifying selection. Colocalization of QTL with differentially expressed genes suggests that polymorphisms in cis-regulatory elements are part of a majority of QTL.

An integrated transcriptomic and comparative genomic analysis of differential gene expression in Arctic charr (Salvelinus alpinus) following seawater exposure.

High-throughput RNA sequencing was used to compare expression profiles in two Arctic charr (Salvelinus alpinus) families post-seawater exposure to identify genes and biological processes involved in hypo-osmoregulation and regulation of salinity tolerance. To further understand the genetic architecture of hypo-osmoregulation, the genomic organization of differentially expressed (DE) genes was also analysed. Using a de novo gill transcriptome assembly we found over 2300 contigs to be DE. Major transporters from the seawater mitochondrion-rich cell (MRC) complex were up-regulated in seawater. Expression ratios for 257 differentially expressed contigs were highly correlated between families, suggesting they are strictly regulated. Based on expression profiles and known molecular pathways we inferred that seawater exposure induced changes in methylation states and elevated peroxynitrite formation in gill. We hypothesized that concomitance between DE immune genes and the transition to a hypo-osmoregulatory state could be related to Cl(-) sequestration by antimicrobial defence mechanisms. Gene ontology analysis revealed that cell division genes were up-regulated, which could reflect the proliferation of ATP1α1b-type seawater MRCs. Comparative genomics analyses suggest that hypo-osmoregulation is influenced by the relative proximities among a contingent of genes on Arctic charr linkage groups AC-4 and AC-12 that exhibit homologous affinities with a region on stickleback chromosome Ga-I. This supports the hypothesis that relative gene location along a chromosome is a property of the genetic architecture of hypo-osmoregulation. Evidence of non-random structure between hypo-osmoregulation candidate genes was found on AC-1/11 and AC-28, suggesting that interchromosomal rearrangements played a role in the evolution of hypo-osmoregulation in Arctic charr.

Genomic arrangement of salinity tolerance QTLs in salmonids: a comparative analysis of Atlantic salmon (Salmo salar) with Arctic charr (Salvelinus alpinus) and rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Quantitative trait locus (QTL) studies show that variation in salinity tolerance in Arctic charr and rainbow trout has a genetic basis, even though both these species have low to moderate salinity tolerance capacities. QTL were observed to localize to homologous linkage group segments within putative chromosomal regions possessing multiple candidate genes. We compared salinity tolerance QTL in rainbow trout and Arctic charr to those detected in a higher salinity tolerant species, Atlantic salmon. The highly derived karyotype of Atlantic salmon allows for the assessment of whether disparity in salinity tolerance in salmonids is associated with differences in genetic architecture. To facilitate these comparisons, we examined the genomic synteny patterns of key candidate genes in the other model teleost fishes that have experienced three whole-genome duplication (3R) events which preceded a fourth (4R) whole genome duplication event common to all salmonid species. RESULTS: Nine linkage groups contained chromosome-wide significant QTL (AS-2, -4p, -4q, -5, -9, -12p, -12q, -14q -17q, -22, and -23), while a single genome-wide significant QTL was located on AS-4q. Salmonid genomes shared the greatest marker homology with the genome of three-spined stickleback. All linkage group arms in Atlantic salmon were syntenic with at least one stickleback chromosome, while 18 arms had multiple affinities. Arm fusions in Atlantic salmon were often between multiple regions bearing salinity tolerance QTL. Nine linkage groups in Arctic charr and six linkage group arms in rainbow trout currently have no synteny alignments with stickleback chromosomes, while eight rainbow trout linkage group arms were syntenic with multiple stickleback chromosomes. Rearrangements in the stickleback lineage involving fusions of ancestral arm segments could account for the 21 chromosome pairs observed in the stickleback karyotype. CONCLUSIONS: Salinity tolerance in salmonids from three genera is to some extent controlled by the same loci. Synteny between QTL in salmonids and candidate genes in stickleback suggests genetic variation at candidate gene loci could affect salinity tolerance in all three salmonids investigated. Candidate genes often occur in pairs on chromosomes, and synteny patterns indicate these pairs are generally conserved in 2R, 3R, and 4R genomes. Synteny maps also suggest that the Atlantic salmon genome contains three larger syntenic combinations of candidate genes that are not evident in any of the other 2R, 3R, or 4R genomes examined. These larger synteny tracts appear to have resulted from ancestral arm fusions that occurred in the Atlantic salmon ancestor. We hypothesize that the superior hypo-osmoregulatory efficiency that is characteristic of Atlantic salmon may be related to these clusters.

The genetic basis of salinity tolerance traits in Arctic charr (Salvelinus alpinus).

BACKGROUND: The capacity to maintain internal ion homeostasis amidst changing conditions is particularly important for teleost fishes whose reproductive cycle is dependent upon movement from freshwater to seawater. Although the physiology of seawater osmoregulation in mitochondria-rich cells of fish gill epithelium is well understood, less is known about the underlying causes of inter- and intraspecific variation in salinity tolerance. We used a genome-scan approach in Arctic charr (Salvelinus alpinus) to map quantitative trait loci (QTL) correlated with variation in four salinity tolerance performance traits and six body size traits. Comparative genomics approaches allowed us to infer whether allelic variation at candidate gene loci (e.g., ATP1α1b, NKCC1, CFTR, and cldn10e) could have underlain observed variation. RESULTS: Combined parental analyses yielded genome-wide significant QTL on linkage groups 8, 14 and 20 for salinity tolerance performance traits, and on 1, 19, 20 and 28 for body size traits. Several QTL exhibited chromosome-wide significance. Among the salinity tolerance performance QTL, trait co-localizations occurred on chromosomes 1, 4, 7, 18 and 20, while the greatest experimental variation was explained by QTL on chromosomes 20 (19.9%), 19 (14.2%), 4 (14.1%) and 12 (13.1%). Several QTL localized to linkage groups exhibiting homeologous affinities, and multiple QTL mapped to regions homologous with the positions of candidate gene loci in other teleosts. There was no gene × environment interaction among body size QTL and ambient salinity. CONCLUSIONS: Variation in salinity tolerance capacity can be mapped to a subset of Arctic charr genomic regions that significantly influence performance in a seawater environment. The detection of QTL on linkage group 12 was consistent with the hypothesis that variation in salinity tolerance may be affected by allelic variation at the ATP1α1b locus. IGF2 may also affect salinity tolerance capacity as suggested by a genome-wide QTL on linkage group 19. The detection of salinity tolerance QTL in homeologous regions suggests that candidate loci duplicated from the salmonid-specific whole-genome duplication may have retained their function on both sets of homeologous chromosomes. Homologous affinities suggest that loci affecting salinity tolerance in Arctic charr may coincide with QTL for smoltification and salinity tolerance traits in rainbow trout. The effects of body size QTL appear to be independent of changes in ambient salinity.

Genome evolution in the fish family salmonidae: generation of a brook charr genetic map and comparisons among charrs (Arctic charr and brook charr) with rainbow trout.

BACKGROUND: Salmonids are regarded as 4R derivative species, having experienced 4 whole genome duplication events in their ancestry. Many duplicated chromosome regions still share extensive homology with one another which is maintained primarily through male-based homeologous chromosome pairings during meiosis. The formation of quadrivalents during meiosis leads to pseudolinkage. This phenomenon is more prevalent within 5 of the 12 ancestral teleost linkage groups in salmonids. RESULTS: We constructed a genetic linkage map for brook charr and used this in combination with the genetic map from Arctic charr, to make comparisons with the genetic map of rainbow trout. Although not all chromosome arms are currently mapped, some homologous chromosome rearrangements were evident between Arctic charr and brook charr. Notably, 10 chromosome arms in brook charr representing 5 metacentric chromosomes in Arctic charr have undergone rearrangements. Three metacentrics have one arm translocated and fused with another chromosome arm in brook charr to a make a new metacentrics while two metacentrics are represented by 4 acrocentric pairs in brook charr. In two cases (i.e., BC-4 and BC-16), an apparent polymorphism was observed with the identification of both a putative metacentric structure (similar to metacentric AC-4 = BC-4 and a joining of acrocentric AC-16 + one arm of AC-28 = BC-16), as well as two separate acrocentric linkage groups evident in the mapping parents. Forty-six of the expected 50 karyotypic arms could be inter-generically assigned. SEX in brook charr (BC-4) was localized to the same homologous linkage group region as in Arctic charr (AC-4). The homeologous affinities detected in the two charr species facilitated the identification of 20 (expected number = 25) shared syntenic regions with rainbow trout, although it is likely that some of these regions were partial or overlapping arm regions. CONCLUSIONS: Inter-generic comparisons among 2 species of charr (genus Salvelinus) and a trout (genus Oncorhynchus) have identified that linkage group arm arrangements are largely retained among these species. Previous studies have revealed that up to 7 regions of high duplicate marker retention occur between Salmo species (i.e., Atlantic salmon and brown trout) and rainbow trout, with 5 of these regions exhibiting higher levels of pseudolinkage. Pseudolinkage was detected in the charr species (i.e., BC-1/21, AC-12/27, AC-6/23, = RT-2p/29q, RT-12p/16p, and RT-27p/31p, respectively) consistent with three of the five 'salmonid-specific' pseudolinkage regions. Chromosome arms with the highest number of duplicated markers in rainbow trout are the linkage group arms with the highest retention of duplicated markers in both charr species.

A 200K SNP chip reveals a novel Pacific salmon louse genotype linked to differential efficacy of emamectin benzoate.

Antiparasitic drugs such as emamectin benzoate (EMB) are relied upon to reduce the parasite load, particularly of the sea louse Lepeophtheirus salmonis, on farmed salmon. The decline in EMB treatment efficacy for this purpose is an important issue for salmon producers around the world, and particularly for those in the Atlantic Ocean where widespread EMB tolerance in sea lice is recognized as a significant problem. Salmon farms in the Northeast Pacific Ocean have not historically experienced the same issues with treatment efficacy, possibly due to the relatively large population of endemic salmonid hosts that serve to both redistribute surviving lice and dilute populations potentially under selection by introducing naïve lice to farms. Frequent migration of lice among farmed and wild hosts should limit the effect of farm-specific selection pressures on changes to the overall allele frequencies of sea lice in the Pacific Ocean. A previous study using microsatellites examined L. salmonis oncorhynchi from 10 Pacific locations from wild and farmed hosts and found no population structure. Recently however, a farm population of sea lice was detected where EMB bioassay exposure tolerance was abnormally elevated. In response, we have developed a Pacific louse draft genome that complements the previously-released Atlantic louse sequence. These genomes were combined with whole-genome re-sequencing data to design a highly sensitive 201,279 marker SNP array applicable for both subspecies (90,827 validated Pacific loci; 153,569 validated Atlantic loci). Notably, kmer spectrum analysis of the re-sequenced samples indicated that Pacific lice exhibit a large within-individual heterozygosity rate (average of 1 in every 72 bases) that is markedly higher than that of Atlantic individuals (1 in every 173 bases). The SNP chip was used to produce a high-density map for Atlantic sea louse linkage group 5 that was previously shown to be associated with EMB tolerance in Atlantic lice. Additionally, 478 Pacific louse samples from farmed and wild hosts obtained between 2005 and 2014 were also genotyped on the array. Clustering analysis allowed us to detect the apparent emergence of an otherwise rare genotype at a high frequency among the lice collected from two farms in 2013 that had reported elevated EMB tolerance. This genotype was not observed in louse samples collected from the same farm in 2010, nor in any lice sampled from other locations prior to 2013. However, this genotype was detected at low frequencies in louse samples from farms in two locations reporting elevated EMB tolerance in 2014. These results suggest that a rare genotype present in Pacific lice may be locally expanded in farms after EMB treatment. Supporting this hypothesis, 437 SNPs associated with this genotype were found to be in a region of linkage group 5 that overlaps the region associated with EMB resistance in Atlantic lice. Finally, five of the top diagnostic SNPs within this region were used to screen lice that had been subjected to an EMB survival assay, revealing a significant association between these SNPs and EMB treatment outcome. To our knowledge this work is the first report to identify a genetic link to altered EMB efficacy in L. salmonis in the Pacific Ocean.

A SNP Based Linkage Map of the Arctic Charr (Salvelinus alpinus) Genome Provides Insights into the Diploidization Process After Whole Genome Duplication.

Diploidization, which follows whole genome duplication events, does not occur evenly across the genome. In salmonid fishes, certain pairs of homeologous chromosomes preserve tetraploid loci in higher frequencies toward the telomeres due to residual tetrasomic inheritance. Research suggests this occurs only in homeologous pairs where one chromosome arm has undergone a fusion event. We present a linkage map for Arctic charr (Salvelinus alpinus), a salmonid species with relatively fewer chromosome fusions. Genotype by sequencing identified 19,418 SNPs, and a linkage map consisting of 4508 markers was constructed from a subset of high quality SNPs and microsatellite markers that were used to anchor the new map to previous versions. Both male- and female-specific linkage maps contained the expected number of 39 linkage groups. The chromosome type associated with each linkage group was determined, and 10 stable metacentric chromosomes were identified, along with a chromosome polymorphism involving the sex chromosome AC04. Two instances of a weak form of pseudolinkage were detected in the telomeric regions of homeologous chromosome arms in both female and male linkage maps. Chromosome arm homologies within the Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) genomes were determined. Paralogous sequence variants (PSVs) were identified, and their comparative BLASTn hit locations showed that duplicate markers exist in higher numbers on seven pairs of homeologous arms, previously identified as preserving tetrasomy in salmonid species. Homeologous arm pairs where neither arm has been part of a fusion event in Arctic charr had fewer PSVs, suggesting faster diploidization rates in these regions.

Integrated (epi)-Genomic Analyses Identify Subgroup-Specific Therapeutic Targets in CNS Rhabdoid Tumors.

We recently reported that atypical teratoid rhabdoid tumors (ATRTs) comprise at least two transcriptional subtypes with different clinical outcomes; however, the mechanisms underlying therapeutic heterogeneity remained unclear. In this study, we analyzed 191 primary ATRTs and 10 ATRT cell lines to define the genomic and epigenomic landscape of ATRTs and identify subgroup-specific therapeutic targets. We found ATRTs segregated into three epigenetic subgroups with distinct genomic profiles, SMARCB1 genotypes, and chromatin landscape that correlated with differential cellular responses to a panel of signaling and epigenetic inhibitors. Significantly, we discovered that differential methylation of a PDGFRB-associated enhancer confers specific sensitivity of group 2 ATRT cells to dasatinib and nilotinib, and suggest that these are promising therapies for this highly lethal ATRT subtype.