Krista M. Nichols

The Genetic Basis of Smoltification-Related Traits in Oncorhynchus mykiss

The timing and propensity for migration between fresh- and seawater is a key theme in the diversity of life histories within the salmonid fishes. Across salmonid species, life-history strategies range from wholly freshwater-resident populations, to migratory and nonmigratory variation within populations, to populations and species that are primarily migratory. Despite the central theme of migration to the evolution of these fishes, the genetic architecture of migration-related processes is poorly understood. Using a genetic cross of clonal lines derived from migratory and nonmigratory life-history types of Onchorhynchus mykiss (steelhead and rainbow trout, respectively), we have dissected the genetic architecture of the complex physiological and morphological transformation that occurs immediately prior to seaward migration (termed smoltification). Quantitative trait loci (QTL) analyses were used to identify the number, effects, and genomic location of loci associated with smoltification-related traits, including growth and condition factor, body coloration, morphology, and osmoregulatory enzymes during the smoltification period. Genetic analyses revealed numerous QTL, but one locus in particular is associated with multiple traits in single and joint analyses. Dissecting the genetic architecture of this highly complex trait has profound implications for understanding the genetic and evolutionary basis of life-history diversity within and among migratory fishes.

Assignment of Rainbow Trout Linkage Groups to Specific Chromosomes

The rainbow trout genetic linkage groups have been assigned to specific chromosomes in the OSU (2N = 60) strain using fluorescence in situ hybridization (FISH) with BAC probes containing genes mapped to each linkage group. There was a rough correlation between chromosome size and size of the genetic linkage map in centimorgans for the genetic maps based on recombination from the female parent. Chromosome size and structure have a major impact on the female:male recombination ratio, which is much higher (up to 10:1 near the centromeres) on the larger metacentric chromosomes compared to smaller acrocentric chromosomes. Eighty percent of the BAC clones containing duplicate genes mapped to a single chromosomal location, suggesting that diploidization resulted in substantial divergence of intergenic regions. The BAC clones that hybridized to both duplicate loci were usually located in the distal portion of the chromosome. Duplicate genes were almost always found at a similar location on the chromosome arm of two different chromosome pairs, suggesting that most of the chromosome rearrangements following tetraploidization were centric fusions and did not involve homeologous chromosomes. The set of BACs compiled for this research will be especially useful in construction of genome maps and identification of QTL for important traits in other salmonid fishes.

Association between DNA polymorphisms tightly linked to MHC class II genes and IHN virus resistance in backcrosses of rainbow and cutthroat trout

Abstract Infectious hematopoietic necrosis (IHN) is a rhabdoviral disease of salmonid species. Single strand conformation polymorphism (SSCP) was used to identify association between DNA polymorphism of the major histocompatibility complex (MHC) class II beta-chain intron and IHN virus (IHNV) resistance in backcrosses of rainbow trout ( Oncorhynchus mykiss ) and cutthroat trout ( O. clarki ). Association was significant ( P =0.024) in four half-sib families from one sire (number 2), but not among half-sibs from another sire (number 1). DNA sequencing analysis revealed that the polymorphism originating from sire 1 is the result of a single base substitution. Sire 2 polymorphism was caused by three different base substitutions. The source of the susceptible allele appears to be from cutthroat trout and not from the rainbow trout population used to produce the hybrids. The results suggest that MHC polymorphism can contribute to linkage and association studies of immunity to infectious diseases in rainbow trout.

Quantitative Trait Loci × Maternal Cytoplasmic Environment Interaction for Development Rate in Oncorhynchus mykiss

Effects of maternal cytoplasmic environment (MCE) on development rate in rainbow trout were evaluated within a quantitative trait loci (QTL) analysis framework. Previous research had identified QTL for development rate in doubled haploid (DH) progeny produced from a cross between the Oregon State University (OSU) and the Swanson (SW) River rainbow trout clonal lines. In this study, progeny for QTL mapping were produced from a cross between the OSU and Clearwater (CW) River clonal lines. Doubled haploids were produced from the OSU × CW F1 by androgenesis using eggs from different females (or MCEs); with androgenesis, the maternal nuclear genome was destroyed by irradiation and diploidy was restored by blocking the first embryonic cleavage by heat shock. All embryos were incubated at the same temperature and development rate quantified as time to hatch. Using a linkage map constructed primarily with AFLP markers, QTL mapping was performed, including MCE covariates and QTL × MCE effects in models for testing. The major QTL for development rate in the OSU×SW cross overlaps with the major QTL found in this OSU × CW cross; effects at this locus were the same across MCEs. Both MCE and QTL × MCE effects contribute to variability in development rate, but QTL × MCE were minor and detected only at small-effect QTL.

Migration‐related phenotypic divergence is associated with epigenetic modifications in rainbow trout

Migration is essential for the reproduction and survival of many animals, yet little is understood about its underlying molecular mechanisms. We used the salmonid Oncorhynchus mykiss to gain mechanistic insight into smoltification, which is a morphological, physiological and behavioural transition undertaken by juveniles in preparation for seaward migration. O. mykiss is experimentally tractable and displays intra‐ and interpopulation variation in migration propensity. Migratory individuals can produce nonmigratory progeny and vice versa, indicating a high degree of phenotypic plasticity. One potential way that phenotypic plasticity might be linked to variation in migration‐related life history tactics is through epigenetic regulation of gene expression. To explore this, we quantitatively measured genome‐scale DNA methylation in fin tissue using reduced representation bisulphite sequencing of F2 siblings produced from a cross between steelhead (migratory) and rainbow trout (nonmigratory) lines. We identified 57 differentially methylated regions (DMRs) between smolt and resident O. mykiss juveniles. DMRs were high in magnitude, with up to 62% differential methylation between life history types, and over half of the gene‐associated DMRs were in transcriptional regulatory regions. Many of the DMRs encode proteins with activity relevant to migration‐related transitions (e.g. circadian rhythm pathway, nervous system development, protein kinase activity). This study provides the first evidence of a relationship between epigenetic variation and life history divergence associated with migration‐related traits in any species.

Quantitative trait loci analyses for meristic traits in Oncorhynchus mykiss

Meristic trait variation among species and populations has long been used as the basis for identification and classification of fishes. Within Oncorhynchus mykiss, there is considerable variation in meristic characters such as numbers of vertebrae, lateral line scales, fin rays, gill rakers, and pyloric caeca. In our laboratory the Oregon State University (OSU) rainbow trout and the Clearwater River (CW) steelhead trout clonal lines, produced by androgenesis, exhibit significant differences in values for meristic traits, making quantitative trait locus (QTL) analysis of these meristic characters possible. Our objective wastodetermine the number, location, and effects of QTL associated with meristic characters in order to test two hypotheses: (1) that QTL for different meristic traits co-localize to the same linkage group and (2) that meristic trait QTL co-localize to the same linkage group as a previously identified development rate QTL. Doubled haploid individuals, produced by androgenesis from sperm from an F1 hybrid between the OSU and CW lines, were used to evaluate the joint segregation of each meristic phenotype and Amplified Fragment Length Polymorphic marker genotypes. Composite interval mapping revealed QTL for six of the seven traits analyzed. One QTL each for scales above the lateral line and for gill rakers co-localized to the same position. Only one QTL for scales above the lateral line co-localized to the same region as that for the development rate QTL, but a greater map resolution is necessary to determine if these loci are truly the same. We failed to detect pleiotropy for most meristic trait QTL. Our results suggest that different major loci are associated with variation in each meristic character and that the expression of these loci may be influenced by maternal and external environmental factors.

Functional Annotation of All Salmonid Genomes (FAASG): An international initiative supporting future salmonid research, conservation and aquaculture

We describe an emerging initiative - the ‘Functional Annotation of All Salmonid Genomes’ (FAASG), which will leverage the extensive trait diversity that has evolved since a whole genome duplication event in the salmonid ancestor, to develop an integrative understanding of the functional genomic basis of phenotypic variation. The outcomes of FAASG will have diverse applications, ranging from improved understanding of genome evolution, to improving the efficiency and sustainability of aquaculture production, supporting the future of fundamental and applied research in an iconic fish lineage of major societal importance.

Examination of reproductive endpoints in goldfish (Carassius auratus) exposed in situ to municipal sewage treatment plant effluent discharges in Michigan, USA

Effects of representative mid-Michigan (USA) wastewater treatment plant (WWTP) effluents on the reproductive physiology of common goldfish (Carassius auratus) were assessed in situ by measuring plasma concentrations of vitellogenin (VTG), 17 beta-estradiol (E2), and testosterone (T), and evaluating gonad histology. Caged adult male and female goldfish were exposed for six weeks to WWTP effluents. One riverine site and one lacustrine site were included as references for comparison to WWTP sites. There was differential growth and gonadal development among locations, which confounded the interpretation of biomarker responses. A linear contrast model was developed by analysis of covariance, and adjusted values were developed for concentrations of VTG, E2, and T in the plasma of both male and female fish. In general, VTG concentrations were significantly less in male than in female goldfish. Most concentrations of VTG in male goldfish were less than the method detection limit. There were no significant differences in plasma VTG of either male or female goldfish among locations or between sites at WWTPs and reference sites. Concentrations of E2 in the plasma of female goldfish were similar among WWTP sites, all of which were less than in females at a pond reference location. Concentrations of E2 in the plasma of male goldfish were similar at all WWTP locations, except for one, where they were greater. No consistent trends in hormone concentrations or gonadal histology could be attributed to putative endocrine disrupter exposure in WWTP effluents. The results indicate that the risk for estrogen agonist exposure below these mid-Michigan WWTPs is small.

Genetic Analysis of Interior Pacific Northwest Oncorhynchus mykiss Reveals Apparent Ancient Hybridization with Westslope Cutthroat Trout

Abstract Seventeen populations of Oncorhynchus mykiss were analyzed for mitochondrial haplotype diversity through either polymerase chain reaction–restriction fragment length polymorphism analysis or mitochondrial DNA sequencing. Six new mitochondrial haplotypes were observed among these populations, five in steelhead and one in Harney Basin rainbow trout. Although no distinct patterns were observed between steelhead and rainbow trout with mitochondrial DNA sequencing, Harney Basin rainbow trout populations from southeastern Oregon exhibited a high frequency of a unique new haplotype designated MYS22. Fourteen of 17 individuals within the Harney Basin rainbow trout populations possessed this haplotype. Additionally, the mitochondrial DNA haplotypes of six westslope cutthroat trout O. clarki lewisi were observed among 90 steelhead from the Tucannon River, Washington, a river in which no native or introduced cutthroat trout have been documented. Analysis of other introduced hatchery and wild stocks of steel...

Evidence of sex-bias in gene expression in the brain transcriptome of two populations of rainbow trout (Oncorhynchus mykiss) with divergent life histories

Sex-bias in gene expression is a mechanism that can generate phenotypic variance between the sexes, however, relatively little is known about how patterns of sex-bias vary during development, and how variable sex-bias is between different populations. To that end, we measured sex-bias in gene expression in the brain transcriptome of rainbow trout (Oncorhynchus mykiss) during the first two years of development. Our sampling included from the fry stage through to when O. mykiss either migrate to the ocean or remain resident and undergo sexual maturation. Samples came from two F1 lines: One from migratory steelhead trout and one from resident rainbow trout. All samples were reared in a common garden environment and RNA sequencing (RNA-seq) was used to estimate patterns of gene expression. A total of 1,716 (4.6% of total) genes showed evidence of sex-bias in gene expression in at least one time point. The majority (96.7%) of sex-biased genes were differentially expressed during the second year of development, indicating that patterns of sex-bias in expression are tied to key developmental events, such as migration and sexual maturation. Mapping of differentially expressed genes to the O. mykiss genome revealed that the X chromosome is enriched for female upregulated genes, and this may indicate a lack of dosage compensation in rainbow trout. There were many more sex-biased genes in the migratory line than the resident line suggesting differences in patterns of gene expression in the brain between populations subjected to different forces of selection. Overall, our results suggest that there is considerable variation in the extent and identity of genes exhibiting sex-bias during the first two years of life. These differentially expressed genes may be connected to developmental differences between the sexes, and/or between adopting a resident or migratory life history.