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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.
MIGRATION is a pervasive theme in the diversification and evolution of animals. Migration has been described as a "syndrome" or a complex suite of integrated physiological, morphological, and behavioral traits that together with environment promotes the drive to move over long distances (Dingle 2006). Across all animal taxa, proximate mechanisms or features such as energetic and morphological changes are intricately tied to the ultimate decision to migrate (Dingle 2006). The genetic architecture of migration and the evolutionary forces that have shaped polyphenism in migration and residency have not been elucidated for any species. In this study, we describe the genetic architecture of proximate physiological and morphological traits associated with migration vs. residency in Oncorhynchus mykiss, a species that consists of both migratory (steelhead trout) and nonmigratory (rainbow trout) life-history types.
Migratory or anadromous salmonids rear in freshwater, migrate to the ocean as juveniles, and return to freshwater to spawn (Groot and Margolis 1991). Prior to seaward migration, juvenile salmonids transform from dark-colored parr, adapted to life in a freshwater stream environment, to silvery smolts, physiologically and morphologically adapted to life in the ocean (Hoar 1976). This physiological transformation, called smoltification, is regulated by environmental cues and complex physiological processes and ultimately culminates in downstream migration (Folmar and Dickhoff 1980; Hoar 1988). Smoltification is generally characterized by an increase in lipid metabolism and protein synthesis, increased growth in length relative to weight, and increased hypo-osmoregulatory ability relative to premigratory and nonmigratory individuals (Folmar and Dickhoff 1980; Hoar 1988; Dickhoff et al. 1997). These processes are modulated by numerous, integrated endocrine changes involving at least three different hormone axes, including the growth hormone, thyroid hormone, and corticosteroid hormone axes (Folmar and Dickhoff 1980; Hoar 1988; Dickhoff et al. 1997). Increased protein synthesis is associated with the deposition of the metabolic by-products guanine and hypoxanthine in the skin and scales of smolts, giving smolts a silvery appearance (Folmar and Dickhoff 1980; Hoar 1988). An increase in growth rate in length relative to weight has profound influences on the body morphology of smolts (Beeman et al. 1994, 1995; Gorbman et al. 1982), rendering the fish more streamlined for a migratory life history. An increase in hypo-osmoregulatory ability is most notably associated with biochemical and morphological changes in the gill and intestines, leading to an increased expression of Na+-K+ ATPase during the transformation (Folmar and Dickhoff 1980; Hoar 1988).
These rapid growth, metabolic, morphological, and behavioral changes in smolts do not occur at the same magnitude in nonmigrant fish. The decision to undergo smoltification and migrate is tightly associated with growth trajectories. Physiological thresholds of body size and growth are often discussed as a mechanism promoting divergence in alternative migratory and nonmigratory life-history trajectories (Thorpe 1994; Dickhoff et al. 1997; Thorpe and Metcalfe 1998; Thorpe et al. 1998). In freshwater, sexual maturation precludes seaward migration (Foote et al. 1994; Thorpe 1994; Thorpe and Metcalfe 1998). Thorpe and others (Thorpe 1994; Thorpe and Metcalfe 1998; Thorpe et al. 1998) have hypothesized that smoltification is a developmental decision made in individuals that have failed to sexually mature in freshwater. Indeed, a negative genetic correlation or genetic trade-off between sexual maturation and smoltification has been observed (Thrower et al. 2004). Although the physiology and the endocrinology of smoltification have been well studied, the ultimate genetic and molecular regulatory mechanisms responsible for alternative migratory and resident life-history types and the genetic trade-offs between these divergent life-history "decisions" are poorly understood. In this study, we test the hypothesis that these multiple physiological and morphological changes associated with smoltification are triggered by one or few major genome regions or that one or few major gene regions work to integrate the complex physiological and morphological changes occurring during smoltification.
The origin of anadromy in salmonid fishes has long been debated (McDowall 1997). Some suggest that anadromy is a derived character arising from wholly freshwater ancestors (Hoar 1976; Stearley 1992). The basis for this theory was originally predicated upon the broad freshwater distribution of salmonid species, parallels among species in the physiological basis of seawater adaptation, and the fact that all species spawn and spend at least part of their life cycle in freshwater (Tchenavin 1939; Quinn and Myers 2004). Phylogenetic relationships among salmonid species constructed from morphological data are in agreement with the "degree" of anadromy. Historically, the mapping of migratory life-history type upon a coarse, morphology-derived phylogenetic tree of salmonids showed a gradation in anadromy—from optionally anadromous (species consisting of either freshwater or anadromous forms) to the most derived obligately anadromous (species consisting exclusively of anadromous forms) (Hoar 1976; Oakley and Phillips 1999). However, more recent molecular Salmonidae and Salmonine phylogenies suggest multiple origins of anadromy when mapped to these trees (Stearley 1992; Stearley and Smith 1993; Oakley and Phillips 1999). On a finer level, observations within species suggest plasticity in the trait and multiple parallel accounts of both the gain and the loss of anadromy in both natural and introduced populations (Quinn et al. 2000, 2001; Unwin et al. 2000; Pascual et al. 2001; Stockwell et al. 2003; Riva-Rossi et al. 2007). While many of the above studies support the theory that timing of and propensity for anadromy is a derived character, having evolved from a freshwater ancestor, alternative views suggest that anadromy is ancestral and that extant salmonids evolved from a marine ancestor (McDowall 1997).
The inheritance of nonmigratory and migratory life-history types and associated traits has been examined in several salmonid species (Refstie et al. 1977; Clarke et al. 1992, 1994; Foote et al. 1992; Johnsson et al. 1994; Thrower et al. 2004; Duston et al. 2005). Although plasticity exists within and across populations (Quinn and Myers 2004; McPhee et al. 2007), inheritance studies suggest that both timing of and propensity for smoltification are also under genetic control. In crosses between life-history variants of Chinook salmon that differ in smoltification timing, Clarke et al. (1994) found that early smolting (smolting during the first year of life, also called "ocean type") was dominant and apparently controlled by few genetic loci; the authors used body morphology and coloration to discriminate between dark, stream-dwelling individuals and silvery, premigratory smolts to determine if Chinook salmon in their study were smolting in their first or second year of life. In species for which both freshwater resident and migratory forms exist, studies evaluating smoltification in intercrosses between resident and anadromous forms indicate that there is also a genetic basis for the propensity to undergo smoltification (Foote et al. 1992; Johnsson et al. 1994). In these studies, hybrid progeny were made from crosses between anadromous sockeye salmon and freshwater resident kokanee (O. nerka) (Foote et al. 1992) and between anadromous steelhead and freshwater resident rainbow trout (O. mykiss) (Johnsson et al. 1994); in both studies, hybrids were intermediate between freshwater and migratory forms in their ability to hypo-osmoregulate in seawater, suggesting that propensity for smoltification is under additive rather than dominant genetic control in these species.
To dissect the genetic basis of alternative migratory and nonmigratory life-history types, we have conducted a comprehensive quantitative trait loci (QTL) analysis of smoltification-related traits in O. mykiss (rainbow and steelhead trout). We evaluate the joint segregation of molecular markers, ordered in the genome, with the segregation of quantitative or polygenic traits associated with smoltification in a cross between nonmigratory rainbow trout and migratory steelhead trout lines. We test the hypotheses that multiple traits are controlled by a few loci of large effect and that multiple, correlated traits of smoltification colocalize to the same regions in the genome.
Genetic crosses:
Genetic analyses of smoltification-related characters were conducted in doubled-haploid progeny produced from a cross between the Oregon State University (OSU) rainbow trout and Clearwater (CW) steelhead trout clonal lines; clonal line origin and production is described elsewhere (Nichols et al. 2007). Briefly, eggs from one OSU female were fertilized with sperm from one CW male to produce a family of F1 progeny. Sperm from the resulting F1 male hybrid clones was used to produce doubled haploid progeny by androgenesis, as described by Young et al. (1998). Doubled haploids were produced in October and reared for ∼3 years at the Washington State University research hatchery. Embryos were maintained in recirculating stack egg incubators at a constant 11° until swim-up (complete yolk absorption). At swim-up, fish were transferred to 5-gallon tanks maintained in a recirculating system at similar densities. At 6 months of age, all fish were transferred to a single 200-gallon tank and were fed by automatic feeder, which was replenished once daily. Temperatures in the recirculating systems ranged from 11° to 18° throughout this study.
Genotypes of doubled-haploid progeny were assessed at multiple loci, using amplified fragment length polymorphic (AFLP), microsatellite, and known gene markers. Dominant AFLP markers are unambiguous in the doubled-haploid progeny, as all individuals are homozygous. Methods for AFLP genotyping are described elsewhere (Nichols et al. 2007). Seventeen AFLP primer sets were used with EcoRI- and MseI-digested genomic DNA. AFLP markers are named according to EcoRI + 3 selective bases, MseI + 3 selective bases, and either an arbitrary number or base pair size of fragment, in that order. Microsatellite markers were chosen on the basis of known placement on existing rainbow trout maps (Sakamoto et al. 2000; Nichols et al. 2003; Danzmann et al. 2005; Guyomard et al. 2006). At least one microsatellite locus per chromosome was chosen to enable the identification of syntenic linkage groups among salmonid linkage maps. Potential candidate genes were chosen on the basis of their putative involvement in the smoltification process, as part of a parallel study aimed to map genes whose products play a role in the endocrine, osmoregulatory, and other physiological changes that occur during smoltification. Primer sets for microsatellite and known genes are provided in supplemental Table S1.
Because smoltification is a temporal process, we chose to sample phenotypes over a time course from February through June 2003 over which the physiological transformation or smoltification was anticipated to occur. In 2003, fish were age 1+, and all fish were sampled again in March 2004 at age 2+ to determine if fish that had not gone through smoltification at 1+ were doing so at age 2+. Since repeated samples or data were taken from the same individuals, fish were sampled nonlethally and with limited invasiveness to minimize handling stress effects. Data on phenotypes were collected February 24, March 17, April 7, April 21, May 5, May 18, and June 9, 2003. At sampling, fish were anesthetized with clove oil and phenotypic data were then collected. Phenotypes collected over this time course included body morphology and skin reflectance from digital photographs, body size (length and weight), gill sodium-potassium ATPase (Na+/K+ ATPase), body condition factor, and instantaneous growth rate in length and weight.
Body morphology:
The change in body morphology from a more deep-bodied, stream-dwelling fish to a more fusiform shape has been well documented in smolting salmonids (Gorbman et al. 1982; Beeman et al. 1994, 1995). We used morphometric shape analysis to quantify variability in body shape among phenotypically divergent doubled-haploid progeny. From digital photographs, 13 landmarks along the left lateral side of the fish were digitized for each time point (Figure 1). Two-dimensional coordinates for landmarks were obtained by digitizing each point using the software tpsDig2 (Rohlf 2005a).
Skin reflectance:
As smolting salmonids are readied for out-migration and adaptation to life in the sea, the metabolic by-products guanine and hypoxanthine are deposited in the skin and scales of smolts, rendering the fish silver in color and highly reflective (Folmar and Dickhoff 1980; Hoar 1988). The concentration of skin guanine has been shown to be highly correlated with color or the reflectance from the skin (Haner et al. 1995). Subjective, qualitative assessments of body silvering have long been used to identify smolts, but more recently, noninvasive measures of skin color or reflectance have been developed to objectively quantify body silver color or reflectance (Duston 1995; Ando et al. 2005). In this study, skin reflectance was quantified by calculating the average pixel intensity within an area directly above the lateral line and below the dorsal fin (Figure 1). To do this, digital photographs were taken in an enclosed box with constant, bright, diffuse light provided by four full-spectrum bulbs. Fish were placed in a shallow, V-shaped aquarium that was affixed with black and white calibration tabs. Digital photographs were taken in this controlled-light environment, using manual camera settings with constant exposure settings for all photos. SigmaScanPro (SysStat Software) software was used to acquire average pixel intensity from this area. Briefly, intensity was calibrated for each image, choosing a two-point calibration: one calibration point on the black tab (scaled at 0) and one calibration point on the white tab (scaled at 100). Using the trace tool, the area mentioned above was defined and calibrated average pixel intensity measured (on a scale from 0 to 100).
Gill Na+/K+ ATPase:
During smoltification, salmonids experience a spike in gill Na+/K+ ATPase, enabling hypo-osmoregulation in seawater (Folmar and Dickhoff 1980; Hoar 1988; McCormick and Bradshaw 2006). At sampling, three to four gill filaments were clipped from each fish nonlethally, placed in 100 μl SEI buffer (150 mm sucrose, 10 mm EDTA, 50 mm imidazole, pH 7.3) at 4°, and then frozen within 4 hr at −80° for storage. Na+/K+ ATPase was quantified in these samples according to McCormick (1993).
Body condition factor:
Condition factor commonly decreases in smolting salmonids relative to nonsmolts (Folmar and Dickhoff 1980; Hoar 1988). Condition factor was quantified using weight (W in grams) and standard length (L in millimeters) with the formula (W/L3 × 100,000).
Growth rates:
Growth rates were calculated from both weights and standard lengths for spring interval from February to June 2003. Relative to nonsmolts, smolts experience a greater increase in growth in length during the spring of smolting (Folmar and Dickhoff 1980; Hoar 1988; Dickhoff et al. 1997). Instantaneous growth rates were calculated using [ln(L2) − ln(L1)]/[t2 − t1] × 100, where L1 and L2 are standard lengths or weights at times 1 and 2 (t1 and t2 in days).
Binary smolt phenotype:
From each sampling point, in addition to the above quantitative data, individuals were scored as smolts (1) or nonsmolts (0) for QTL analyses. The binary smolt phenotype was assigned on the basis of the digital photographs taken for reflectance and morphology. Individuals were categorized as smolts if they were streamlined, reflective, silvery individuals characteristic of this life-history type. Individuals with distinctive parr marks along the lateral line through the duration of the study are typical of stream-dwelling parr and were categorized as nonsmolts. Individuals that were more deeply bodied, yellow, and spotted in color are more characteristic of larger freshwater-dwelling rainbow trout and were also categorized as nonsmolts. Fish that could not be reliably categorized were scored as missing or unknown.
Sex phenotype:
The phenotypic sex of the fish surviving to March 2004 was determined by internal examination of gonads. Fish were euthanized with a lethal dose of anesthetic (clove oil) prior to dissection. For summary statistical analyses, males were coded as 1 and females as 0.
Statistical analyses:
Body morphology, thin plate spline analysis:
Morphometric shape analysis was conducted using a thin plate spline analysis, using tpsRelw software (Rohlf 2005b). Landmarks from all fish from all time points were superimposed to calculate the generalized least-squares Procrustes average or consensus shape, eliminating size differences (Rohlf and Slice 1990). From the consensus shape, each individual sampled at each time point was evaluated for deformation from the consensus shape, and variation captured in partial warp scores (Rohlf and Bookstein 2003). The covariance matrix of partial warp scores for all individuals at all time points was used in a principal-components analysis to distill partial warp scores into fewer orthogonal dimensions describing variation in shape (Bookstein 1991). Principal components of partial warp scores are called relative warps, and these relative warps were taken as metrics for shape for use in QTL analyses.
All traits, summary statistics:
Traits were organized over the time course of sampling. With longitudinal sampling of each individual, one could conceivably conduct genotype–phenotype analyses of the trajectories of individual traits using fitting of orthogonal polynomials (Yang et al. 2006). However, because these methods are not readily implemented for interval mapping in non-growth-related traits, we chose to use metrics that were informative on whether or not the fish were smolting during the time course of the spring, based upon our biological knowledge of the physiological and morphological changes that occur during smoltification. Using time-course data, we chose to use (1) the minimum condition factor, since smolts have lower condition factors than nonsmolts; (2) the maximum and empirical range of skin reflectance values, since smolts will have larger values and may change most dramatically during smoltification; (3) the maximum Na+/K+ATPase value, since smolts achieve a higher peak ATPase during smoltification, returning to baseline levels if they do not migrate to the ocean; (4) maximum values of relative warps obtained from thin plate spline analyses (along six orthogonal axes); and (5) spring growth rates (taken as the growth from February to June) in length and weight. Individuals with incomplete time-course data were given missing data values for QTL analyses. Using the metrics chosen for each trait above, data were tested for outliers and assumptions of normality and homogeneity of variance prior to QTL analyses. Data that were nonnormal in distribution were transformed to achieve normality. Correlations between trait values used in QTL analyses were conducted using Pearson's correlations in the case where data were normal and Spearman's correlations in the case where data were not normally distributed. One-way analysis of variance was conducted (PROC GLM; SAS Statistical Software, Cary, NC) to examine whether means for continuous traits were significantly different between the sexes and life-history categories. The model used to test the null hypothesis that the means for each trait () were the same for males and females () was , where is the phenotype of individual j of sex i. For tests of whether continuous-trait means were the same for the binary life-history category (LHbin), or for smolts and nonsmolts (), the model was used, where is the continuous-trait phenotype of individual j with LHbin category i. Finally, to test whether continuous-trait means were significantly different among the life-history categories (LHcat), smolt, rainbow, parr, and unknowns (), the model was evaluated. When overall models were significant, Tukey–Kramer tests were used to determine which pairs of means were significantly different. In all tests, statistical significance was determined with a type I error rate of α = 0.05.
Genetic analyses:
Linkage map construction:
Prior to linkage map construction, markers were evaluated for segregation distortion, or deviation from the expected 1:1 genotype ratio in doubled-haploid progeny. Markers showing significant distortion were checked for reliability and genotyping errors and were removed from the analysis if unreliable. Only markers for which >80% of individuals were genotyped were used in subsequent analyses. The genetic linkage map was constructed using Mapmaker/EXP (Lander et al. 1987) as described by Nichols et al. (2007).
QTL analyses:
Quantitative trait loci analyses were conducted using interval and composite-interval mapping (Zeng 1994) in R/qtl (Broman et al. 2003); multiple-trait interval mapping (Kao et al. 1999) was conducted using QTL Cartographer (Basten et al. 1994, 2004). In all analyses, permutation tests (Churchill and Doerge 1994; Doerge and Churchill 1996) were performed (n = 1000 permutations for each test) to determine the threshold for log of the odds (LOD) or likelihood-ratio (LR) scores, using a type I error rate of α = 0.05. To control the overall familywise error rate (FWER) in the case of multiple-QTL analyses for single traits, the highest value of the 95% LOD thresholds for all single-trait interval-mapping permutation tests was chosen as the cutoff for inclusion of QTL in the second step of composite-interval mapping, which is outlined below. This is more conservative than using false discovery rate (FDR) methods for simple interval mapping (Benjamini and Yekutieli 2005). The issue of controlling for multiple tests for subsequent single-trait composite-interval mapping (CIM) becomes more difficult, since the models for each QTL scan in CIM are different for each trait; in this case, choosing an overall FWER might be too stringent if chosen among models that range from the inclusion of one additional background cofactor to multiple background cofactors. In this case, significant QTL identified in step 2 below were those that exceeded the 95% permutation threshold for that trait.
Single-trait interval mapping:
R/qtl does not include a model for doubled haploids; thus a backcross model coding for the doubled haploids was used since models for both cross types utilize two genotypic classes for tests of significant QTL effects. In the first step, single QTL were identified with interval mapping using the EM algorithm in R, scanning for QTL at marker positions and every 2 cM in the genome. In this first interval-mapping step, the model (model 1) was used test the null hypotheses that QTL additive effects are zero (), where is the phenotype for individual with marker genotype ; and is the marker or calculated QTL genotype (given flanking marker genotypes), where = 0 for the OSU genotype and = 1 for the CW genotype. In the second step, significant QTL, identified in the first step, were used as cofactors in a search for additional QTL in the genome, using the model (model 2), testing again for significant QTL effects () at every 2-cM position. All terms in model 2 are the same as described above, and for the OSU genotype and for the CW genotype at the significant QTL positions identified in model 1. New QTL identified in model 2 were added to the set of QTL, and model 2 was tested again at every 2-cM position in the genome until no new QTL were identified in the context of this multiple-QTL model. Two-locus epistasis was tested at all markers and at every 5-cM position in the genome; here, epistasis was tested comparing the likelihood of the model that includes an interaction between two QTL positions ( and ), + (model 3), to the likelihood of the model including only main effects (where ). QTL positions were refined with all significant terms (single and interacting QTL) included in the model, as described by Nichols et al. (2007). Briefly, refining the QTL position involved the use of model 2 above, with the addition of significant interacting QTL from tests of model 3; refining the position of each QTL was accomplished by iteratively testing the likelihood of each QTL position within a chromosome in the context of the multiple-QTL model, until the most likely position of each QTL remained unchanged. Once positions were refined, QTL effects and percentage of variation explained (PVE) were estimated in the context of the multiple-QTL model, using a drop-one-term analysis as described by Nichols et al. (2007). Two-LOD support intervals were estimated for each identified QTL and have been shown to be conservative estimates of 95% confidence intervals for QTL position (Visscher et al. 1996).
Multiple-trait interval mapping:
Multiple-trait QTL mapping was performed using interval mapping with a doubled-haploid design in QTL Cartographer (Basten et al. 2004). Multiple-trait analyses were performed (a) with all traits used to quantify migration-related physiology and morphology and (b) with traits that colocalized to QTL in the genome. Multiple-trait permutation tests were performed by shuffling traits for each individual as a block, maintaining the correlation structure among traits while shuffling traits across marker genotypes.
Phenotypic traits:
A single family of 110 doubled-haploid individuals was measured for physiological and morphological traits related to smoltification. Phenotypic trait values used in QTL analyses are summarized below by life-history category assigned to each fish. Of the 110 fish used for QTL analyses, 22 (20%) were categorized as smolts, 17 (15%) as parr, and 23 (21%) as rainbow; 48 fish (44%) were intermediate in phenotype and were scored as unknowns at age 1+. Continuous-trait data are summarized below by life-history category (including unknowns) to illustrate the differences among the life-history types used to categorize the binary trait. For the binary trait, all smolts were categorized as "smolts," while parr and rainbow were categorized as "nonsmolts"; intermediate fish were treated as missing data for the binary trait.
Phenotypes used for QTL analyses and summarized below include maximum relative warp values for six axes describing body morphology (maxrelw1–maxrelw6), maximum and range of skin reflectance values (maxrefl and rangerefl, respectively), maximum gill Na+/K+ ATPase (natural log-transformed, ln_maxatpase), minimum condition factor (minkfact), growth in standard length and weight from January through June (sprgrsl and sprgrwt, respectively), binary smolt phenotype (binary), and sex phenotype (sex). With the exception of categorical traits (sex and binary phenotype) and gill Na+/K+ ATPase, all traits conformed to the assumptions of normality and homogeneity of variance.
Body morphology:
Thirteen landmarks digitized along the left side of the fish (Figure 1) revealed significant variation in body morphology (Figure 2). Thin plate spline analyses of all doubled-haploid progeny at all time points (n = 677) revealed that the first six relative warps explained 77% of the variation in body shape; each of these relative warps explained from 5% (relw6) to 24% (relw1) of the variation in shape. Some individuals or time points were missing from the analysis due either to mortality before the end of the study or to poor-quality photographs for accurate digitization of landmarks. Examination of the shape variability captured by each of the relative warps (Figure 2) reveals that this analysis has readily captured quantitative shape variability within the doubled-haploid family. Much of the variability, looking at the extreme values for each relative warp in relation to the consensus (Figure 2), appears to be differences in (1) dorso-ventral body depth (landmarks 3, 4, and 11), (2) snout morphology (landmarks 1, 2, 12, and 13), and (3) caudal peduncle depth and length (landmarks 5–9). Relative warp 1 explained 24% of variation in shape; although rainbow trout appear to have lower maximum relw1 (Figure 3A), the differences among categories are not statistically significant [F = 2.43, d.f. = 3, P = 0.0719 (categorical); F = 0.6, d.f. = 1, P = 0.4437 (binary)]. Relative warp 2 accounted for 17% of shape variation. Fish with an undetermined life-history category had the greatest maximum relw2 values, while rainbow trout had the smallest (Figure 3A). Unknowns were significantly different from rainbow for maxrelw2, but no other pairs were statistically significant (F = 5.42, d.f. = 3, P = 0.0020). When life-history type is coded as a binary trait, not surprisingly from the similar means among smolt, parr, and rainbow, there was no statistical difference between smolts and nonsmolts for maxrelw2 (F = 0.34, d.f. = 1, P = 0.5648). Relative warp 3 explained 12% of the variation in morphology and most obviously captured the shape differences among migratory and nonmigratory life-history types (Figure 3A). Relative warp 3 appears to explain differences in dorso-ventral body depth and in caudal peduncle length and depth (Figure 2). Rainbow trout had lower average maximum relw3 (were more deep bodied), while smolts had larger maximum relw3 values (were more fusiform or less deep bodied). Parr and intermediates were morphologically similar to each other for maxrelw3, but were significantly different from smolts and rainbow trout (F = 15.54, d.f. = 3, P < 0.0001). When coded as a binary trait, nonsmolts and smolts were statistically different from one another for maxrelw3 (F = 15.52, d.f. = 1, P = 0.0003). Relative warp 4 accounted for 10% of shape variation. Maximum relw4 values were on average lower for parr than for all other groups (Figure 3A, F = 4.53, d.f. = 3, P = 0.0057). This statistical difference among groups was not observed when life-history category was coded as a binary trait (F = 0.44, d.f. = 1, P = 0.5111). Relative warps 5 and 6 explained 9 and 5% of shape variation, respectively. Neither maximum relw5 nor relw6 showed significant variation among life-history types, irrespective of coding.
Skin reflectance:
Average pixel intensity of the area below the dorsal fin (Figure 1) provided a quantitative estimate of the degree of reflectance or silvery color of each fish. Both maximum (maxrefl: F = 17.32, d.f. = 3, P < 0.0001) and range of (rangerefl: F = 6.73, d.f. = 3, P = 0.0004) reflectance showed significant differences among life-history groups (Figure 3B). Reflectance values were also significantly different when life-history types were encoded as a binary trait (maxrefl: F = 37.16, d.f. = 1, P < 0.0001) (rangerefl: F = 14.82, d.f. = 1, P = 0.0003). Compared to nonsmolts and unknowns, smolts had a significantly greater average maximum reflectance (were more reflective or silver in color; Figure 3B). Smolts also had a greater range in reflectance values during the time course of sampling, when compared to unknowns and rainbow trout, but not when compared to parr (Figure 3B).
Gill Na+/K+ ATPase:
Na+/K+ ATPase values were natural-log transformed to achieve normality. ATPase values were significantly different among life-history categories (F = 4.12, d.f. = 3, P = 0.0086; Figure 3C), but only marginally significant when evaluated for binary encoded life-history types (F = 3.95, d.f. = 1, P = 0.0520). Smolts had greater average maximum ATPase than unknowns and parr, but were not significantly different from the rainbow trout life-history type (Figure 3C).
Body condition factor:
Body condition factor calculates the relative contribution of weight to length. For a given length, fish with greater weight will have a larger condition factor. Minimum body condition factor was greater on average for the rainbow trout life-history type than for all other life-history types that were similar in average minimum condition factor (Figure 3D, F = 8.28, d.f. = 3, P < 0.0001). When compared using binary trait categories, the same trend is observed: nonsmolts (which include rainbow trout) have a significantly greater condition factor than smolts (F = 9.81, d.f. = 1, P = 0.0028).
Growth rates:
Spring growth rates in standard length and weight were not significantly different among life-history types (Figure 3E).
Binary smolt phenotype:
Sixty-two individuals (56%) were scored for binary smolt phenotype. Of the individuals scored, 22 individuals (35%) were categorized as smolts, and 40 (65%) were categorized as nonsmolts. In the nonsmolt category, 17 fish (42.5% of nonsmolts and 27% of all scored) were categorized as smaller parr, while 23 fish (57.5% of nonsmolts and 37% of all scored) were categorized as larger rainbow trout. The 44% of individuals that were not scored were intermediate in their phenotypes and difficult to categorize as either a smolt or a nonsmolt, and were categorized as unknowns for summary statistical analyses.
Sex phenotype:
Of the 110 individuals sampled for smoltification, 74 individuals (67%) survived to the next year when phenotypic sex was determined by internal examination of gonads. Of those sexed, 49% (n = 36) were male and 51% were female (n = 38). Sex significantly contributed to variation in both maximum reflectance (maxrefl, F = 5.41, P = 0.02) and gill Na+/K+ ATPase (maxatpase, F = 5.60, P = 0.02), but not to any other traits (data not shown). Inclusion of sex as a cofactor in QTL analyses for these two traits did not significantly affect the detection of QTL (data not shown).
Correlation among traits:
All traits used in QTL analyses were correlated with at least one other trait, with the exception of the sixth axis of morphometric shape (maxrelw6) (Table 1). Correlations between traits that were alternative expressions of reflectance [maximum and range (maxrefl, rangerefl), r = 0.942, P < 0.0001] and of growth in standard length and weight (sprgrsl and sprgrwt, r = 0.606, P < 0.0001) were greatest (Table 1). Fish with the greatest maximum reflectance also had a larger range in reflectance. Both of these metrics of reflectance were correlated with the binary smolt phenotype; smolts tended to have both higher maxima and greater ranges in reflectance. Reflectance was correlated to a lesser degree with body morphology. Maximum reflectance was negatively correlated with maxrelw2, for which no QTL were identified. Reflectance range was negatively correlated with maxrelw4, for which a QTL was colocalized with most other smoltification-related traits. Finally, the range but not the maximum reflectance had a negative correlation with minimum condition factor. This indicates that fish with a greater change in body coloration also had the lowest condition factor, which is consistent with long-standing observations that smolts both change in body coloration with smoltification and have a reduced condition factor relative to nonsmolts and the presmolting stage. Not surprisingly, fish with greater growth in standard length also experienced greater growth in weight, but this relationship is weaker than that observed for range of and maximum reflectance. Growth in both standard length and weight was negatively correlated with relative warp 3 (maxrelw3) and positively correlated with condition factor (minkfact). Individuals with lower growth rates in both length and weight generally had higher maxrelw3 values (were more fusiform in shape) and lower minimum condition factors. At the same time, individuals with higher growth rates in length and weight had lower maxrelw3 values (were more deep bodied) and higher minimum condition factors. Additional axes of body morphology, aside from those mentioned above, were also correlated with minimum condition factor. The first three relative warps were negatively correlated with condition factor; individuals with higher values for these relative warps generally had lower minimum condition factors. The binary smolt phenotype was positively correlated with reflectance (both maximum and range) and maxrelw3 and negatively correlated with minimum condition factor. Individuals that were scored as smolts had greater maximum and range of reflectance, greater values for maxrelw3 (more fusiform in shape), and lower minimum condition factors.
Genetic linkage map:
Twenty-nine major linkage groups were identified with linkage analysis in 110 doubled-haploid individuals, using 263 markers and the sex phenotype. The number of major groups corresponds to the number of chromosome pairs revealed by karyotyping in the OSU and CW clonal lines and hybrids (Phillips et al. 2005). Synteny among linkage groups in this study and other rainbow trout maps (Sakamoto et al. 2000; Nichols et al. 2003; Danzmann et al. 2005; Guyomard et al. 2006) is established by mapped syntenic microsatellite markers; linkage groups are named according to the cross (OC) and the linkage group number defined in prior maps (Figure 4; only groups with significant QTL are shown). The average intermarker distance within linkage groups, including markers with 0-cM distances, was 5.8 cM; excluding markers that were completely linked (0-cM distance), the average intermarker distance was 7.5 cM. The sex phenotype mapped to linkage group OC1 and was perfectly linked to marker Ssa408UoS (0-cM distance). This map was the framework for our QTL analysis of smoltification-related traits.
QTL analyses:
With composite-interval mapping for single traits, QTL were identified for each trait with the exception of morphology metrics maxrelw2 and maxrelw6 and gill Na+/K+ ATPase (Figure 4, Table 2). QTL are named according to the trait and the linkage group to which they localized. In all cases, the genotypic effect of a QTL refers to the estimated phenotypic difference between the CW genotype and the OSU genotype attributed to the additive effects of alleles at that QTL. A positive genotypic effect would indicate that individuals with a CW genotype at a particular QTL would have a greater phenotypic trait value, attributed to that locus, than individuals with the OSU genotype. Note that although determining statistical thresholds for significance in the context of multiple tests is not a simple one to address in the context of IM and CIM for the detection of QTL, most all QTL, in the context of the multiple-QTL models, were significant at P < 0.0001. Although a Bonferroni correction for the 13 single traits may not be correct in the case of multiple nonindependent traits, even in the most conservative case that the traits are independent, the nominal P-value for significance would be α = 0.05/13 or 0.004. All except for one QTL for body morphology (maxrelw3-OC8) were significantly below this threshold in the multiple-QTL models.
Body morphology:
Ten QTL for maximum relative warp values for four axes of shape were identified (Figure 4 and Table 2). For maxrelw1, 2 QTL were identified on two linkage groups, OC13 and OC31, explaining 11.6 and 18.5% of the phenotypic variation in maxrelw1. The QTL on linkage group OC31 colocalizes with QTL for maxrelw5, another axis of morphometric shape. No QTL were identified for maxrelw2, despite the significant variation observed among the fish sampled. For maxrelw3, the descriptor that captured dorso-ventral variability in body depth and caudal peduncle shape, 4 main-effect QTL were identified. These QTL explained 3.0–14.1% of the phenotypic variability along this shape axis. The QTL on linkage group OC8 was statistically significant in a single-QTL interval-mapping scan, but did not significantly contribute to the overall, multiple-QTL model when all other QTL were included (Table 2). Maxrelw3 QTL on linkage groups OC8 and OC20 colocalized with QTL for other traits in this analysis. Each of the 4 QTL for maxrelw3 had a positive genotypic effect, indicating that individuals with a CW genotype (paternal) at these loci had larger maximum values of relw3 than those with an OSU genotype at this locus; this difference in genotypic values at these loci exhibits a more dorso-ventrally flattened or fusiform fish, characteristic of smolts, in those with the CW genotype relative to those with the OSU genotype. For maxrelw4, 3 QTL were identified, explaining 10.4–18.1% of variation in shape along this axis. The two QTL with the greatest effects were found on OC9 and OC20, and both had negative genotypic effects. The QTL for maxrelw4 on OC20 (maxrelw4-OC20) colocalized to the same region as maxrelw3-OC20 and other smoltification traits. One additional QTL was identified for body morphology with maxrelw5. The QTL for this morphological shape descriptor explained 14.1% of shape variation and was localized to OC31, the same region as that for maxrelw1-OC31.
Skin reflectance:
A total of four QTL were identified for the two metrics of skin reflectance, maximum reflectance (maxrefl) and range of reflectance (rangerefl) (Table 2, Figure 4). Two QTL were detected for rangerefl, rangerefl-OC16 and rangerefl-OC20; these QTL accounted for 11.2 and 24.3% of the variation in the range of reflectance, respectively. Two QTL were identified for maxrefl, maxrefl-OC17 and maxrefl-OC20, which explained 14.4 and 11.1% of the variation in maximum reflectance, respectively. QTL for reflectance on OC16 and OC17 did not colocalize with QTL for other traits; however, reflectance QTL on OC20 did colocalize with QTL for other traits on this linkage group (Figure 4). Each of the reflectance QTL had positive genotypic effects (Table 2), indicating that individuals with a CW genotype at each locus had a greater maximum or range of reflectance than individuals with an OSU genotype.
Gill Na+/K+ ATPase:
No QTL were detected for maximum log-transformed gill Na+/K+ ATPase, despite the variation observed among life-history types.
Body condition factor:
One QTL for minimum body condition factor (minkfact) was identified on OC20 (Table 2); this QTL colocalizes to the same region as many other traits, including reflectance and body morphology QTL (Figure 4). The minkfact-OC20 QTL accounted for 13.8% of the variation in minimum condition factor and had a negative genotypic effect. Individuals with a CW genotype at this locus had a smaller minimum condition factor compared to those with an OSU genotype.
Growth rate:
QTL for spring growth rate in standard length (sprgrsl) and weight (sprgrwt) colocalized to linkage groups OC8 and OC20 (Table 2). It is clear that sprgrsl-OC8 and sprgrwt-OC8 colocalize to the same region, but 2-LOD support intervals for sprgrsl-OC20 and sprgrwt-OC20 do not overlap on linkage group OC20 (Figure 4). One additional QTL (sprgrsl-OC14) and two-locus epistasis were revealed for spring growth rate in standard length (Table 2). Main-effect QTL for spring growth rate in standard length each had positive genotypic effects; individuals with a CW genotype had greater spring growth rate in standard length compared to those with an OSU genotype with differences ranging from 0.025 ± 0.017% (sprgrsl-OC20) to 0.060 ± 0.014% (sprgrsl-OC8) growth in standard length per day (Table 2). Epistasis between sprgrsl-OC14 and sprgrsl-OC20 had the opposite effect on growth rate; individuals with CW genotypes at both of these loci had 0.140 ± 0.034% lower growth rate in standard length than individuals with OSU genotypes at both loci (Figure 5). The two QTL for spring growth in weight were opposite in their effects. For sprgrwt-OC8, individuals with a CW genotype had larger growth rates in weight relative to those with an OSU genotype (genotypic effect: 0.172 ± 0.044% growth in weight per day); for sprgrwt-OC20, individuals with a CW genotype exhibited a slower spring growth rate in weight compared to those with the OSU genotype (genotypic effect: −0.174 ± 0.044% growth in weight per day).
Binary smolt phenotype:
Two main-effect QTL were identified for the binary smolt phenotype (Figure 4). These QTL were detected on OC6 and OC20 and explained 9.0 and 8.8%, respectively, of the variation in the binary phenotype (Table 2). In a linear model or analysis of variance context, like that employed in the drop-one-term analysis, main-effect regression coefficients from binary trait data are estimates of the penetrance of the loci identified (Coffman et al. 2005). Therefore, the genotypic effects (Table 2) exhibit the penetrance differences for OSU and CW genotypes for each QTL, in the context of a multiple-binary-trait loci model. A positive genotypic effect in this case indicates that the CW genotype is more penetrant; in other words, individuals with the CW genotype are more likely to be smolts than individuals with the OSU genotype at a particular locus. For simplicity, we plot observed proportions of smolts and nonsmolts for each genotype at markers most closely linked to the identified binary trait loci (Figure 6, A–C). For binary_smolt-OC20, the CW genotype was more penetrant than the OSU genotype; 82% of those classified as smolts possessed the CW genotype at binary_smolt-OC20 (and scored for the binary phenotype), while only 18% of smolts possessed the OSU genotype at this locus (Figure 6A). Oppositely, binary_smolt-OC6 had a greater penetrance in those with the OSU genotype; a greater proportion of individuals with the OSU genotype at this locus were smolts compared to those with the CW genotype. For binary_smolt-OC6, 64% of those classified as smolts possessed the OSU genotype, while only 36% of smolting individuals possessed the CW genotype at this locus (Figure 6B). Epistasis significantly contributed to the variation in binary phenotype; binary_smolt-OC14 and binary_smolt-OC25 showed a significant interactive effect, explaining 13.6% of the variation in binary phenotype (Table 2), but did not affect variation in penetrance as main-effect QTL. The LOD scores for these individual loci appear significant (Table 2), but simply reflect the significance of dropping main-effect and all higher-order interactions including that locus; for these loci, composite-interval mapping including all other significant terms as cofactors does not identify main-effect QTL at these loci (data not shown). Complete penetrance (100%, n = 11/11) was observed as a result of epistasis between OSU genotypes at binary_smolt-OC14 and binary_smolt-OC25; on the other hand, no individuals with CW and OSU genotypes at binary_smolt-OC14 and binary_smolt-OC25, respectively, were smolts (n = 0/11; Figure 6C). The remaining two joint genotypic classes produced intermediate proportions of smolts: 25% (n = 4/12) of individuals with CW, CW genotypes and 36% (n = 5/14) with OSU, CW genotypes at binary_smolt-OC14 and binary_smolt-OC25 were smolts (Figure 6C).
Multiple-trait analysis:
Joint- or multiple-trait QTL analyses were conducted using (a) all 12 traits used to quantify physiological and morphological metrics of smoltification and (b) only the 7 quantitative traits that colocalized to OC20 (underlined in Table 1). For each of these scenarios, correlations were not significant for all pairs of traits, but each trait was significantly correlated with at least one other trait (Table 1), with the exception of maxrelw6. A single-QTL interval-mapping scan for all 12 traits revealed a total of 4 QTL (Figure 4). Approximate P-values for significance were obtained from 1000 permutations of the phenotypes (shuffled as a block) across genotypes. Joint QTL for the 12-trait data set were found on linkage groups OC8 (LOD = 10.45, P < 0.01 from 1000 permutations), OC14 (LOD = 9.32, 0.01 < P < 0.05), OC20 (LOD = 16.70, P < 0.01), and OC31 (LOD = 9.02, 0.01 < P < 0.05). When the 12-trait joint QTL on OC20 was included as a background cofactor in the model, all main-effect QTL remained significant except the QTL on OC14 (LOD = 1.96, P ≫ 0.05); no additional main-effect QTL were identified. Joint QTL for the 7-trait data set (including traits colocalizing to OC20) also revealed 4 QTL, but not all in the same positions as those identified in the 12-trait data set (Figure 4). Joint QTL for the 7-trait data set were found on linkage groups OC8 (LOD = 7.35, P < 0.01), OC20 (LOD = 13.35, P < 0.01), OC21 (LOD = 7.09, P < 0.01), and OC30 (LOD = 7.73, P < 0.01). All joint QTL in the 7-trait data set remained significant when the QTL on OC20 was included as a background cofactor in a genome scan, and no additional joint QTL were identified. Not surprisingly, joint QTL for both analyses colocalize to regions where multiple single traits localize, most notably for OC-8 and OC-20 (Figure 4).
We have identified several genome regions associated with variation in multiple morphological and physiological indexes of smoltification in O. mykiss, a species that consists of both resident and anadromous or migratory life-history types. This is the first study to reveal genomic loci associated with migration physiology in any fish. Our results provide a foundation for a deeper understanding of the genes and regulatory mechanisms underlying smoltification, or the decision to migrate or stay, in this species. As we reveal the genomic regions and genes involved in this important life-history decision, we may begin to understand the evolutionary history of smoltification within O. mykiss and gain a better understanding of how genes and environment influence the decision to migrate or maintain residency. Ultimately, this study lays the foundation for parallel studies in this and other salmonid species to begin asking questions about the origins and evolution of migration vs. residency.
Our findings indicate that a few genomic loci contribute to variation in the individual trait phenotypes and that one genome region is strongly associated with multiple smoltification traits. The distribution of QTL effects in this study is not unlike the skewed distribution of QTL effects in QTL analyses of complex traits observed across many taxa, and with the limitations of sample size in this study, it is likely that small-effect loci were not detected (Lynch and Walsh 1998; Roff 2007). The fact that all traits for which significant differences were observed among life-history types are associated with a common genome region suggests that either a single locus or multiple linked loci may play a role in the physiology associated with migration vs. residency. The QTL on OC20 are associated with body morphology, skin coloration or silvering, condition factor, and growth rate; not surprisingly, when these traits are considered together either subjectively in binary trait analysis or objectively in joint analysis, the same major region is identified. The fact that multiple single-trait QTL underlie the binary smoltification QTL further corroborates the idea that migration vs. residency is a threshold trait, regulated by underlying continuous traits that contribute to the decision to migrate or stay. Does the colocalization of continuous and binary traits suggest that a master regulatory region or gene may play a significant role in the integrated cascade of events that promote smoltification or residency? It cannot be ruled out that a single gene upstream of the complex, integrated endocrine and growth cascades is responsible for regulating the "switch" that determines whether an individual will undergo smoltification. Despite the long history of studies on smolt physiology, no studies have yet detected the molecular processes triggering and integrating these complex endocrine and physiological processes involved in smoltification. Dissection of the genome region on OC20 together with functional studies will provide clues on whether a single trigger controls many of the physiological differences between smolts and residents.
For most of the detected QTL in this study, the majority of additive effects are unidirectional for individual quantitative traits. For example, for traits significantly segregating between smolt and nonsmolt categories (relative warps 3 and 4, reflectance (maximum and range), and minimum condition factor) there is a preponderance of genotypic effects with the same sign or influencing the trait in the same direction. Under neutral expectations, the signs or directions of effects are expected to be random for a given phenotype (Orr 1998). Our results suggest that additive effects for proximate physiology associated with smolt status may be a result of directional selection. In other words, genes associated with smoltification physiology and morphology are primarily found only in the steelhead line (CW). This result, however, does not hold for the binary-trait QTL. In binary-trait analysis, one locus, found on the linkage group where most other quantitative traits localized (OC20), influences the trait as expected under directional selection and fixation of steelhead alleles in the steelhead line (and nonsmolt alleles in the rainbow trout line). However, the other binary trait locus detected (on OC6) has an opposite effect; individuals possessing the steelhead (CW) allele at this locus have a lower propensity for smolting than OSU. Taken together, there is clear evidence that the multiple genes for being steelhead or rainbow trout in smoltification physiology have been fixed for the most part in the OSU and CW lines, but there is still some evidence for "rainbow trout" genes in the steelhead line and vice versa. What does this mean for adaptive genetic variation for smoltification in the populations from which the lines were derived? Depending on allele frequencies in the source populations, this may mean that even where one life-history strategy is predominant, there may be adaptive potential to respond to environmental change.
In this study, we revealed genomic loci associated with life-history diversity in an evolutionarily divergent cross of O. mykiss (between a rainbow trout derived from California and a steelhead trout derived from Idaho), and it is unknown whether loci associated with smoltification in this wide cross would be the same between sympatric O. mykiss life-history types. Across the natural range of O. mykiss, there is considerable variability in the propensity to migrate. Landlocked and the most northerly populations are wholly resident, while populations with connections to the sea exhibit variable proportions of migratory individuals ranging from wholly anadromous to partially anadromous (a mixture of anadromous and resident life-history types) (Quinn and Myers 2004; McPhee et al. 2007). The degree of genetic differentiation between resident and anadromous populations of O. mykiss in sympatry is variable. Most studies examining genetic differentiation between sympatric life-history types of O. mykiss reveal that alternative life-history forms within the same freshwater system are more closely related to one another than are the same life-history types across river systems (Docker and Heath 2003; Narum et al. 2004; Olsen et al. 2006; McPhee et al. 2007); however, there are some instances where significant reproductive isolation has been observed between sympatric O. mykiss life-history forms (Docker and Heath 2003; Narum et al. 2004). In other words, resident and anadromous O. mykiss in sympatry exhibit some level of gene flow, and there is evidence for plasticity in the expression of the alternative life histories. The close genetic relationship between resident and anadromous life-history types in sympatry is not unique to O. mykiss, but is found in other salmonid species that exhibit variation in anadromy vs. residency (Hindar et al. 1991; Jones et al. 1997; Quinn 2005). With possible multiple parallel examples of life-history diversification within and across salmonid species, the question remains whether the same or different sets of genes play a role in the diversification of (1) anadromous and resident salmonid life histories and (2) timing of migration in anadromous species. Continued studies examining the genetic architecture of these alternative life-history strategies within and among populations and species will address these questions.
The salmonid lineage experienced a whole-genome duplication by autotetraploidization ∼25–100 MYA (Allendorf and Thorgaard 1984). The recent genome duplication within salmonids affords the opportunity to ask questions regarding how duplicated genome regions play a role in the evolution of complex phenotypes. Genomic resources for the salmonid lineage are more extensive than for any other major group of fishes and provide the opportunity to understand complex trait evolution following a genome duplication in multiple, closely related species. Homeologous relationships among duplicated chromosome arms have been identified for several salmonid species (Danzmann et al. 2005; Gharbi et al. 2006; Guyomard et al. 2006). In our study, we find direct evidence that at least one pair of duplicated, homeologous genome regions is associated with smoltification-related traits. The duplicated EST-linked microsatellite, OMM5017 is linked to a QTL identified in the 12-trait joint analysis and to a lesser degree with QTL for spring growth rate in standard length on homeologous linkage groups OC14 and OC20 (Figure 4). For homeologous chromosomes (for which >1 marker has previously identified homeologues) from prior O. mykiss mapping studies (Nichols et al. 2003; Danzmann et al. 2005; Guyomard et al. 2006), there is indirect evidence that QTL for body morphology (maxrelw4) localize to homeologous linkage groups OC9 and OC20. We cannot ascertain whether the duplicate genes or closely linked loci are fundamentally associated with smoltification trait variation in this study. However, this and other studies (O'Malley et al. 2002; Moghadam et al. 2007) suggest that duplicate copies could play a role in the expression of quantitative traits in salmonid taxa. The role of duplication and subsequent duplicate gene evolution remains an important question as the genetic architecture of life histories and other quantitative traits is revealed in this major group of fishes.
To summarize, we have identified genome regions associated with smoltification or the physiological and morphological transition that occurs prior to seaward migration. In particular, one region that is associated with multiple traits lends insight into the genetic basis of multiple correlated traits. We emphasize that in the absence of functional tests for migratory displacement, we cannot ascertain whether genome regions associated with the ultimate decision to migrate are the same as that associated with these proximate metrics of smoltification. The question of whether this same region is associated with smoltification in other salmonids remains unanswered, but this study lays the foundation for parallel studies in this and other species. Ultimately, with parallel accounts of the genetic architecture of smoltification and migration in multiple salmonid species, we can begin to understand the evolution of anadromy on both coarse and fine scales.
The authors thank Kyle Sundin, Kyle Martin, Steve Patton, Alan Inderrieden, and Lee Suksdorf for assistance in fish sampling at Washington State University. Trout eggs for the production of clonal lines and doubled haploids were provided by Troutlodge. Bench space for ATPase quantification was kindly provided by Penny Swanson and Brian Beckman, Northwest Fisheries Science Center (NWFSC), and laboratory assistance was provided by Kathy Cooper. Philip Haner was helpful in discussions on quantification of body reflectance. The authors thank Gary Winans and James Rohlf for discussions on morphometric statistical analyses. Later molecular work was completed at the NWFSC in the Conservation Biology DNA laboratory under the direction of Linda Park and Michael Ford, with the kind assistance of Elizabeth Heeg. Julia Sharp, Montana State University, was helpful with general statistical discussions. Rodney McPhail, Purdue University, was helpful in the production of scientific illustrations and figures. Karl Broman, Johns Hopkins University, kindly provided code for refining QTL positions in R/qtl and was helpful in discussions on statistical issues in QTL analyses. The authors thank Michael Ford and Ted Morgan and anonymous reviewers and the editor for comments that improved the final manuscript. This research was funded by the National Science Foundation to G.H.T. (IBN-0082773) and by a National Research Council postdoctoral fellowship to K.M.N.
Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. EU084717, EU084718, EU084727, EU084728, EU084733, EU084734, EU084737, EU084738, and EU084741–EU084744.
↵2 Present address: Instituto de Acuicultura de Torre de la Sal (IATS), Consejo Superior de Investigaciones Científicas (CSIC), 12595 Castellón, Spain.
Communicating editor: L. McIntyre
- Received November 6, 2007.
- Accepted May 1, 2008.
- Copyright © 2008 by the Genetics Society of America


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Senior Backend Engineer, Finance Squad
Software Engineering - Paris - Full-time
We believe in a world where all cars are shared. Carsharing empowers people to get going in a smarter, easier way, while also having a positive impact on the environment and making cities more liveable. It's this vision that propels us forward and inspires us to think even bigger.
Getaround is the world's leading carsharing platform with a community of more than 5 million users sharing thousands of car in more than 300 cities in the U.S and Europe.
Our team is collaborative, positive, curious, and engaged. We think fast, work smart, laugh often, and are looking for like-minded people to join us in our mission to disrupt car ownership and make cities better.
Your mission:
- The product & engineering team is getting close to 50 people in Europe, but we have a lot of positions open and we will grow a lot more this year. To scale effectively, we organize ourselves in squads with people coming from various departments like engineering, data, design, and product.
- Fulfill Getaround's financial obligations and build a scalable and easy-to-use accounting system.
- Empower other squads with easy to use abstractions to grow our business.
Getaround is a marketplace collecting Millions of euros per month on behalf of our owners, drivers and insurance partners. The finance squad has 2 main missions:
What you'll work on:
- In practical terms, your work will be very diverse and will span all over the codebase, you can have a look at this blog entry where Nicolas explains what his job is like in the finance squad:
- Define and implement long term changes to support growth and product iterations, like going from only supporting one time payments to subscriptions, supporting different payment methods or adding pre-authorization for risky behaviors.
- Shape our codebase to gracefully handle regulatory challenges, like the PCI-DSS compliance or the recently released PSD2 aiming at securing online payments with two-factor authentication.
- Produce automated reporting for the finance department to use. Seems boring, isn't it? Actually the needs of a finance department are driving a lot of fun constraints to play with; continually increasing volumes, consistency, reliability, and precision (to the last penny!).
- Supporting other squads; Most squads need at some point something from the finance squad. It could be some assistance for a given task, or it could be a feature that isn't yet supported and that we'll need to add to the components we own.
- Develop the vision of the squad using continuous refactoring to build a more decoupled architecture and simple-to-use abstractions to be used by other developers.
Who you'll work with:
- You'll be working with David and Nicolas, Backend Engineers, and Lucian, product owner of the squad, but will be in contact with all the other squads when needed. You will report to Michael.
What you'll bring to the table:
- You've shipped and maintained code in production
- Good knowledge of at least one backend language
- Experience with a relational database such as PostgreSQL or MySQL
- You're a rigorous engineer that relish creating highly reliable backend services
- You have a good understanding of system design, data structures, and architecture
- You care about quality and you know what it means to ship high-quality code
- Good communication and teamwork skills
- Able to communicate in English
Our Tech Stack:
- Ruby on Rails 5.2 with Ruby 2.6
- TypeScript, React/Preact with Webpack
- Continuous integration with Docker and CircleCI to push to production multiple times a day
- MySQL, Redis, Snowflake
- A strong internal design system
- Monitoring dashboards built with Telegraf + Influxdb + Grafana
What we offer:
- A solid engineering team with a lot of experience
- The peace of mind of a large test suite and pull requests to stay sane and ship often
- Getting to learn from your peers and to share your knowledge on the blog and in our internal presentations every two weeks
- A ticket to one technical conference of your choice each year
- Open source projects are being extracted from the codebase when appropriate
- We'll buy any non fiction books you want to read, and you'll get access to our growing library
- An international startup environment with 20+ different nationalities, HQ in San Francisco and European offices in Paris, Berlin, Barcelona, London, Amsterdam, Oslo and Nantes
- A remote friendly culture, with some remote allowed for this position
- We often organise meetups in our office
- Tickets restaurants, a good health insurance
- "Hack days" to experiment with new technologies and ideas as well as other team events
No need for a resume, but Linkedin, Twitter and Github usernames are appreciated! Don't hesitate to give our backend challenge a try.
Come join us in continuing to make Getaround a great place to work!

subset: commoncrawl


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Know more about this business than we do? Cool! Please submit any corrections or missing details you may have.Help us make it right
Harmony Motor Home Service located at 8001 SE Harmony Road in Portland, OR services vehicles for AC and Heating Repair, Mufflers Repair. Call (503) 654-0674 to book an appointment or to hear more about the services of Harmony Motor Home Service.
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The mechanic took over 5 months to repair my car. I had to constantly call to check on his progress. He also quoted me $450 to repair it when he supposedly found out what the problem was. It turned out that there was something else wrong and had a specialist come in to find the problem. He finally called last week and wanted to charge me more money and also informed me that there was also a problem with the alternator. When I asked him why I should pay him more when he quoted me $450 and took over 5 months to fix the care, his answer was that I told him to take his time. Then he continued to insult me about my profession by saying "I wish all I had to do for a living is pound nails into boards" and told me I was the worst customer he ever had. Hows that for customer service.
Harmony Auto Service Inc was founded in 1971, and is located at 8001 Se Harmony Rd in Milwaukie. It employs 2 employees and is generating approximately $160,000.00 in annual revenue.
Harmony Auto Service is located at 8001 SE Harmony Rd, Milwaukie, OR. This location is in the Southgate neighborhood. This business specializes in RV Repair & Service.


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Berlin's House of One will bring Jews, Christians, and Muslims together. Each religion will have its own special section of the building, and they can come together in a communal room in the middle.
The worship center will be built where Berlin's first church once stood, 800 years ago, and is meant to reflect Berlin's multiculturalism and diversity.
Story by Jacob Shamsian and editing by Alana Yzola


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The Bruce Murray Space Image Library is a unique collection – recent and past photos and videos from the world's space agencies, artwork, diagrams, and amateur-processed space images. Bruce Murray, Planetary Society cofounder and emeritus director of NASA's Jet Propulsion Laboratory, insisted that planetary missions take photos for the public as well as for scientists. Bruce literally helped change our picture of the solar system.
Total Solar Eclipse Montage
Total solar ecilpse
Transit of the Sun by Deimos, Curiosity sol 2350
Transit of the Sun by Phobos, Curiosity sol 2359
Totality at 38,000 feet
Eye safety during an eclipse
Partial solar eclipse of 9 May 2013 as viewed from Hawaii
Geometry of a total solar eclipse
Solar chromosphere and diamond ring near totality
The November 3, 2013 solar eclipse in Africa seen from space (video)
Annular eclipse of the Sun by Phobos, Curiosity sol 363
May 10, 2013 solar eclipse from Russian satellite Elektro-L
May 10, 2013 solar eclipse viewed by Elektro-L
Mars moon Phobos eclipse
Hinode views an annular solar eclipse, January 4, 2010
Hinode views January 4, 2010 annular solar eclipse
SDO observes its first lunar transit
2010 Total Solar Eclipse
A solar eclipse - as viewed from the Moon
Annular eclipse, May 20, 2012
SDO observes a lunar transit of the Sun


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Passenger Reservation System in Indian Railways
Adequate safeguards have been kept in the computerised Passenger Reservation System (PRS) and internet booking system to prevent its misuse by unscrupulous elements.
For the benefit of different categories of travelling public, the Railways have introduced ticketing facilities at Non-Railhead locations and India Post Offices so that the ticketing system can be accessed by people living in smaller towns and villages. This is in addition to e-ticketing which provides access to reservation system from the comfort of the home/office.
In addition to the provisions made in the computerised PRS and internet ticketing system to prevent misuse of reserved ticketing system, various other steps have been taken, some of which are as under: (i) Condition of carrying original proof of identity by any one passenger booked on a reserved ticket. (ii) Agents are debarred from booking tickets during first thirty minutes of opening of booking, i.e., from 0800 to 0830 hours for general booking, 1000 to 1030 hours and 1100 to 1130 hours for Tatkal booking on AC class and Non-AC class respectively. (iii) In case of booking of reserved tickets through internet, only one booking in one user login session except for return/onward journey between 0800 and 1200 hours. (iv) The monthly limit of booking of tickets through internet by an individual user has now been revised to 6 tickets in a month instead of 10 tickets.
This Press Release based on information given by the Minister of State for Railways Shri Manoj Sinha in a written reply to a question in Lok Sabha on 27.04.2016 (Wednesday).


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• 1 Object of agreement
4yourfairs acts only as an intermediary and provides private apartments and single-family homes as fair accommodation between tenants and landlords. The accommodation contract exists directly between the tenant and the landlord. The landlord is taken in the 4yourfairs database free of charge. A claim for successful mediation does not arise as a result. After a lease request of a potential tenant, 4yourfairs will clarify the current vacancy with the landlord. The landlord may request a deletion from the database at any time. However, already mediated bookings still have to be implemented as agreed.
• 2 Booking procedure
After his request, the tenant gets a non-binding offer from 4yourfairs. In case of any special requirements (allergies etc.) the tenant needs to inform 4yourfairs in writing in advance. After the assumption of the offer by the tenant and the final check of the vacancy with the landlord, 4yourfairs draws up an accommodation contract between the tenant and the landlord. The mediation is considered as successfully completed after tenant and landlord both agreed to the offer. Both parties will get a copy of the signed contract. In addition the tenant signs a commission contract with 4yourfairs.
• 3 Payment and commission
The amount reported in the offer is a final price including the commision fee. As soon as the accommodation agreement has been accepted by the tenant and the landlord, the tenant receives an invoice concerning the agency fee of 4yourfairs. The payment of this fee has to be paid directly to the account of 4yourfairs, cashless via bank transfer. The claim to commission remains the same even in later reductions of the rental fee, for examle due to cancellations. The payment of the rental fee has to be transfered by the tenant directly to the account of the landlord 10 days before arrival. For short-term bookings, the payment will be adjusted accordingly. Any transfer costs are to be paid by the tenant.
• 4 Check in
The owner is obliged to take the guests mediated by 4yourfairs on arrival and to hand over the key of the house or apartment. The landlord guarantees the exclusive occupancy of the property by the tenant. The time of the handover of the rental will be agreed directly between the landlord and the tenant. 4yourfairs will provide the contact data to both accordingly
• 5 Obligations of the landlord
The guests are to be accommodated in the private rooms, shown on the photos provided by the landlord. The entire accommodation has to be handed over in a clean and proper condition. If breakfast is included, the ingredients are to be provided in accordance with the tenant. The guest beds are freshly made, towels are provided by the landlord in sufficient quantity. On arrival and for the entire rental period, the landlord has to be reached by phone for the tenant and 4yourfairs.
• 6 Obligations of the tenant
The overnight guests are obliged to treat the property and the objects in it carefully and return them in the same condition as found. Any damage to the property or equipment is to be reported to the owner immediately.
• 7 Contract Changes
A change request by the tenant after signing the accommodation contract is to be addressed to 4yourfairs as soon as possible. This includes in particular the announcement of additional overnight guests and changes concerning the rental period. 4yourfairs will inform the landlord in order to obtain the consent. Only after the explizit approval of the landlord the contract will actually change. In case the amendment causes an increase of the rent, it affects equally the commission. In this case the tenant receives a revised invoice from 4yourfairs. In case of a minority occupancy or a reduction of the rental period after signing the contract, the rent and the appropriate commission is not to be reduced or refunded. All changes after signing the contract have to be announced in writing.
• 8 Cancelation conditions
After consent of booking by mail or contract the tenant remains the right to withdraw from the booking. The cancelation must be in writing form and shall be addressed directly to the landlord with a copy to 4yourfairs. The following cancelation fees apply:
up to 61 days before arrival = 20% of the agreed rental fee
60 to 35 days before arrival = 50% of the agreed rental fee
from 34 days before arrival = 80% of the agreed rental fee
no-show on the arrival date = 100% of the agreed rental fee
In case of a cancellation the commission claim of 4yourfairs remains unchanged.
After accepting the booking the landlord can only cancel the agreement for serious reasons, like unpredictable damages to the accommodation which make the rental impossible. In case of a cancellation forced by the landlord the landlord has no claim for the rental fee and 4yourfairs may charge him the commission for findig a new accommodation for the tenant. In addition the landlord has to pay possible additional costs incurred to the tenant through the cancellation and the rental of the substitute accommodation.
Any cancellation has to be sent in written form directly to the contract partners with a copy to 4yourfairs.
• 9 Compensation
If the landlord does not provide the fair accommodation to the tenant on arrival or should the accommodation not be available due to a reason represented by the landlord, the tenant is entitled to compensation. Any defect on the property is to be communicated to the landlord immediately, so that he will be given the opportunity to remedy. The tenant is entitled to a reduction of the rent, if the landlord fails to meet its obligations.
• 10 Change of guests / higher or lower occupancy
If a change of guest(s) during the rental period is desired, the feasibility is to be agreed directly between tenant and landlord. If possible, the landlord will clean the room for the new guest, prepare the bed again and hand out fresh towels. For these additional services a rate will be charged. The tenant informs the overnight guests about the fact that non-agreed person represents a change of contract that entitles the owner to the appropriate recalculation. Short term unoccupied rooms are to be fully paid by the tenant.
• 11 Visual material
Photographic material provided by the landlord remains his property and is used by 4yourfairs exclusively for offers to potential tenants. The rights for visual material taken by 4yourfairs remains with 4yourfairs.
• 12 Protection clause
Tenant and landlord guarantee towards 4yourfairs not to enter within the next four year after departute into a direct liaision of accommodation. For any non-compliance with this clause, the lost commission is to paid to 4yourfairs.
• 13 Liability
As an intermediary 4yourfairs is only liable for the activities of the agency. The liability is limited to gross negligence and intent. Against 4yourfairs no claims for damages can be made if the contract should not be respected. Furthermore 4yourfairs is not liable for any misleading or false information about the rental and for losses arising from a breach of contract by the tenant or the landlord. 4yourfairs is not liable for any damages of asset, property and/or personal injury caused to the landlord by the tenant or to the tenant by the landlord. The tenant is responsible for all the people he has placed in the rental. He is liable for any damages caused in or on the accommodation and property of the landlord.
as of 01.04.2019


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So this is "Delinquent Pair," a dramatic machinima with a lot of interesting stuff going on. I appear first in Episode 3 as Alex, but I took over for another voice actor so the same character appears in the previous episodes (below).
But if you just wanna jump in and listen to me, I appear first at 17:34 and then pretty much stick around for the rest of the episode. I'm the one in the red armour. ;D
The previous episodes are here:


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Parental Liability for the Acts of a Minor Child
Is a parent responsible for the tortious acts of their minor child? Generally, a parent does not have any liability for a tort committed by their child. However, there a few limited exceptions.
Parental Liability for a Minor's Driving in California
Since, in California, the parent or legal guardian must sign a driver's license application for any minor who is under 18, section 17707 essentially spells out civil liability for that parent or guardian if the minor causes a car accident which provides: "Any civil liability of a minor arising out of his driving a motor vehicle…is hereby imposed upon the person who signed and verified the application of the minor for a license."
Agent and Principal
A parent is liable for the acts committed by their child when that child is acting within the course and scope of a relationship built on an agency theory. The best example of something like this is when a parent asks their child to go to the store to pick something up for them. The child at that instance is under the control and direction of their parent. As such the parent is responsible for the child as an employer is for an employee.
Knowledge of Prior Misconduct
A parent may sometimes be considered liable for an injury caused by their own child where the parent's own negligence made it possible for the child to cause the injury.
In other words, the evidence must show that the parents had a duty as a reasonable person to restrain the child. This is a fact intensive inquiry that is generally not easily answered. The evidence must demonstrate that the parent knew or should have known that their child could have committed the type of act which injured the individual. If for example a minor has a history of painting graffiti a future act of tagging using the parents paint maybe foreseeable.
Statutory Liability
Statutory liability exists in certain situations where the child engages in "willful misconduct." Cal. Civil Code § 1741.1. This statute does not impute liability for ordinary negligence but where the act rises above negligence. The damages that can be awarded under this statute are also capped at $ 25,000.00.
The statute imposes liability on parents where bodily injury happens as the result of the child's willful misconduct that results in physical harm.
Interesting Lawsuit of the Month: Woman Charged in Alleged FAKE McDonald's Coffee Burn Case
Does this sound familiar?- a woman claims that an unsecured lid caused steaming hot McDonald's coffee to spill on her right hand, severely burning it. As "evidence", Selena Edwards, 38, of Victorville provided pictures of second-degree burns.
BUT the only burns Edwards may suffer from are the prosecutorial ones she now faces for allegedly faking her injuries.
State insurance officials say the woman's claims were a bid to extract $10,000 from McDonald's Corp. The photos and medical documents Edwards provided to bolster her case came from the Internet, they said.
"We discovered that some of the photos were from a hospital website," state Insurance Commissioner Dave Jones said. "We contacted her medical provider and discovered she hadn't received any medical treatment."
The San Bernardino County district attorney charged Edwards with 21 felony counts of insurance fraud and workers' compensation fraud.
Authorities said Edwards looked to the golden arches in Fontana, to allegedly commit the crime. Officials said the Victorville woman claimed coffee had spilled on her right hand when she was handed a cup with an unsecured lid at the McDonald's drive-through.
"By copying legitimate burn photos from the Internet, Edwards attempted to make a profit from another person's pain and suffering and for this she will be prosecuted to the full extent of the law," he said.
The prosecution of Edwards comes 20 years after a jury awarded $2.9 million to a 79-year-old woman who was badly burned after hot coffee spilled into her lap at a McDonald's in Albuquerque. The 1994 verdict attracted international attention, was mocked by radio and television talk-show hosts and was even used as a plot point in the TV comedy "Seinfeld."
Despite the controversy, the woman in that case, Stella Liebeck, actually did suffer severe third-degree burns and required skin graft surgery.
Jones said that every year tens of thousands of cases that potentially involve fraud are referred to his agency.
Quote of the Month
"No matter what, expect the unexpected. And whenever possible BE the unexpected." – Lynda Barry, author


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A very informative article was recently published outlining one author's success self-publishing over traditional publishing, most notably in terms of higher net royalties on book sales. In fact, the case study recorded significantly higher royalties on a lower quantity of book sales along that self-publishing route.
The book pricing advantages of self publishing is no stranger to this blog, nor the increasingly successful population of authors who follow that path. But this particular article also mentioned that writers should never have to pay for publishing upfront.
Unfortunately, I've seen authors who have been pulled in by that concept, but end up publishing an often poorly produced book sold back to them at highly marked-up costs. (Publishers are businesses and need to make money, after all.) So that model really only puts poorly produced books right back in the hands of authors, not readers.
The successful alternative does involve upfront publishing fees, which opens a direct contract between authors and publishers including quality, professional production on books that are competitively sold in the marketplace, where readers buy books. Make sure your self-publishing choice includes those things like cover design, interior formatting, and full distribution. Also, as I've mentioned before – and the significance here is worth the redundancy – make sure your publisher offers pricing flexibility (control) and 100% royalties on book sales.
I hope that helps. Have fun and keep writing…
Karl Schroeder


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About Weight Loss Surgery Price In Madison
Diet recommendations after gastric sleeve vary depending on where the surgery is performed and your individual situation. A gastric sleeve diet generally follows a staged approach to assist you ease back into eating solid foods as you recuperate. How rapidly you move from one period to the next depends on how rapidly your body heals and adapts to the change in eating patterns. You can normally begin eating regular foods about three months after the surgery. You should drink enough fluids after the bariatric surgery to avoid dehydration.
Sanatoriums that attain a minimum of 375 bariatric surgeries per annum has the superb safe registers while those that have achieved below 75 per annum possess the finest degree of difficult situations appearing as demonstrated by the research done by a hospital evaluation society. The outstanding sanatorium with the very best lap band surgeon will remove all the possible problems like inside hemorrhage of blood, malfunctioning lung or demise. This surgeons are perfect and in advance they'll perform an evaluation to decide if the bariatric surgery is suitable and later on they'll select the correct technique for you.
Before opting for a weight loss surgery, there are specific requirements that you will need determined by the surgical process of your choice. For example gastric sleeve demands comprise things like inability to reduce and maintain weight through diet and exercise, a BMI of 40 and above and a complex case of obesity. Before going for these surgeries, you need to see an expert so that you can see if there may be a few other weight loss alternatives because operations are generally complicated along with the very fact that some are permanent or involve adding foreign objects in the body makes them the last option as far as you weight loss is concerned.
Although lap awful surgery has functioned for thousands of folks around the world, this bariatric surgery process has some disadvantages that may make it your last pick for weight solution. Not only is it slow in giving results, it is also quite complicated consequently it raises the odds of you being re-ran. To add on this, it calls for a foreign item that is the silicon band to remain within your body , which may become quite uncomfortable at times. As much as the silicon band will be inside your body already, it may take a long time before the body begins to react to its effects.
Bariatric surgery is the best treatment for severe obesity, it creates dramatic and durable weight loss surgery consequences. It's unusually safe for large patients but it's some associated negative side effects. Just as other weight loss surgeries, bariatric surgery also introduce potential health risks both in the short term and in the long run. This dangers include excessive bleeding, infection, adverse reactions to anesthesia, blood clots, breathing difficulties, leaks in your gastrointestinal system and passing in very rare cases. More duration hazards and complications vary depending on the specific kind of weight loss surgery.
Madison, WI Weather
Helpful Search Links


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FLINT -- A concern over possible E. coli contamination in ground beef products prompted all Halo Burger restaurants to close Sunday as a precautionary measure.
The restaurants were reopened Monday after the local chain, in cooperation with its meat supplier Flint-based Abbott's Meat Inc., got new shipments of ground beef from a Detroit company, Terry Thomas, chief executive officer for Flint-based Halo Burger, said today.
"All of us are very concerned and never want to get anything out there where the public would be affected. We don't think they will be affected. I think everything was stopped in time," Thomas said.
Thomas said his company got a call Saturday night from Abbott's President Edward Abbott about the possible contamination.
Abbott's supplies the meat for all 11 Halo Burger restaurants and has done so for the past four or five years. Only two restaurants were open at the time of the call, Thomas said.
"I immediately ceased operations," Thomas said. "I told (my son) to shut down the stores. We kept the stores closed on Sunday."
Thomas said Abbott's done extra testing of all meat it receives and was told the meat sent out had been tested.
The problem was discovered through a routine microbiological testing by the U.S. Department of Agriculture's Food Safety and Inspection Service.
"I've been through their plant on a couple occasions," Thomas said.
"When the meat comes into their plant they spend (extra money) to do another test on the product, which is not required of them. For something like this to happen to (Abbott's) is absolutely unbelievable."Thomas said even if there was any E. coli in the meat they received, the Halo Burger restaurants normally cook all their beef at 250 degrees, which would kill the E. coli. But even though the meat probably was safe, Thomas said, they took no chances and got rid off all the beef they had and replaced it.
Thomas said they've received no complaints from people becoming ill.
"I question it (government's findings), but once the government says something there's nothing you can do about it," he said.
The meat to Halo Burger was among 26,669 pounds of ground beef being voluntarily recalled by Abbott's.
The USDA department has not received any reports of illnesses associated with consumption of these products.
The recalled meat includes pounds of coney island topping and topping mix as well as pounds of ground beef patties.
It was not immediately known what area businesses or restaurants may have or had the meat being recalled. A news release from the company stated the meat was sold to hotels, restaurants and other businesses in the Flint, Saginaw, Bay City, Midland, Lapeer, Lansing and surrounding areas.
A spokeswoman for Abbott's said the company was not releasing the names of the impacted businesses.
In a news release, Abbott's President Edward Abbott said, "Our No. 1 concern is for public safety. Always has been, always will be. This is the only E. coli recall in our 100-year history.
"We do not believe the contamination originated in our handling and packaging of the products. However, we do feel it is necessary to do everything in our power to expedite the recall process."
Abbott could not be reached for additional comment this morning.
The ground beef products were produced between July 12 and July 20 and were distributed to hotels, restaurants and institutions in Michigan, according to the USDA.
Anyone with questions about the recall can contact Abbott's Meat at (810) 232-7128.
FLINT -- A concern over possible E. coli contamination in ground beef products prompted all Halo Burger restaurants to close Sunday as a precautionary measure.
Note to readers: if you purchase something through one of our affiliate links we may earn a commission.


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Wednesday, December 14, 2011
Wine from a prestigious estate in the heart of Bordeaux and a three Michelin-starred chef come with very high expectations and of course, a very high price tag. I signed up for a dinner at the Leela Palace Hotel in Bengaluru recently and expected to be blown away that evening. I have to remind you all that Alain Passard owns and operates the restaurant Arpege in Paris which has three Michelin stars and is VEGETARIAN!
The reception at the Leela was regal and warm. We were offered Champagne and vegetarian Hors d'oeuvres at the Lobby bar where we had the opportunity to meet and greet the other guests for the evening. A little while later, we were led across the porch of Leela to the Banquet hall with rose petals strewn over us.
We were seated in predetermined tables and given some time to say hello to the other occupants of the table and get to know each other before we moved on to the five course dinner.
We were greeted by Mr.Sanjay Menon who is a successful wine importer and educator from Mumbai, followed by the CEO of Chateau Margaux. We were to taste 5 different wines from different years along with the five courses of our dinner.
White Arlequin in Himalayan honey and candied lemon
*Pavillon blanc 2009
Tomato and Blackberry "a la fourchette"
*Pavillon rouge 2004
Red roasted Onion in hibiscus sauce, red carrot Orange mousseline
*Chateau Margaux 1999
This dish was undoubtedly the star of the evening. It tasted amazingly good and went to show how a Michelin starred chef can turn a humble onion into a hero. No wonder I licked my plate clean! All you Onion-haters out there, you are missing out on something BIG!!!
Celerisotto with black Perigord Truffle
*Chateau Margaux 1989
Unlike what we expected from the name of the dish, this course did not have any rice in the risotto. It was a risotto made with finely chopped celeriac in a creamy sauce and shaved truffle on top. I have to admit that the flavour of the truffle was very mild and disappointing. A good drizzle of truffle oil would have done a much better job in adding flavour to the dish instead of the fresh truffle on top.
Avocado souffle with chocolate and pistachio
*Chateau Margaux 2003
The final and least impressive of the courses was the dessert. The organisers did mention that they were having trouble with getting the 70 odd souffles set on time to be served to the guests at the same time. But that does not justify serving a dessert that was not completely set. Also the flavour of the pistachio was over-powering and left me rather disappointed.
Overall, the food and wine at the dinner did not meet my expectations. But the evening on its own, was a wonderful experience. I had the privilege of sitting next to a famous and award-winning chef and also a talented writer was seated in our table along with some of my dear friends. Would I sign up for another evening like this? Yes, I would.
Posted by Divya Chandran at 10:21 PM No comments:
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