Journal of Heredity 2005:96(2):97–107
doi:10.1093/jhered/esi019
Advance Access publication January 13, 2005
ª The American Genetic Association. 2005. All rights reserved.
For Permissions, please email: [email protected]
Sex-linked Quantitative Trait Loci for
Thermotolerance and Length in the
Rainbow Trout
G. M. L. PERRY, M. M. FERGUSON, T. SAKAMOTO,
AND
R. G. DANZMANN
From the Groupe interuniversitaire de recherches océanographiques du Québec (GIROQ), Département de biologie,
Université Laval, Ste-Foy, Québec G1K 7P4, Canada (Perry); Department of Zoology, University of Guelph, Guelph,
Ontario N1G 2W1, Canada (Ferguson, and Danzmann); and Tokyo University of Marine Science and Technology, Faculty of
Marine Science, Department of Marine Biosciences, 4–5–7, Konan, Minato, Tokyo 108-0075, Japan (Sakamoto).
Address correspondence to G. M. L. Perry at the above address, or email: [email protected].
Abstract
We hypothesized that correlation between growth traits and upper thermal tolerance (UTT) in rainbow trout (Oncorhynchus
mykiss) might be explained by quantitative trait loci (QTL) localized to the same linkage groups. Microsatellites on three
autosomal linkage groups carrying UTT QTL in rainbow trout were tested for associations with fork length (FL) and
condition factor (K) in half-sib families of outbred rainbow trout and in backcrosses of trout lines selected on UTT.
Additionally, we used a sex-linked microsatellite (OmyFGT19TUF) to test for marker-trait associations at the sex
chromosomes. The sex-linked marker OmyFGT19TUF was significantly associated with FL and UTT, accounting for up to
9.6% and 9.7% of variance in these traits, respectively. Male advantages in FL (and, to a lesser extent, UTT) relative to their
female sibs were dependent on the origin of the Y chromosome and thus varied among grandsire lines. However, males had
higher K in a manner unrelated to Y chromosomal origin, suggesting a partially sex-limited expression of this trait.
Omy325UoG was significantly associated with K in one of the outbred half-sib families, but no other significant autosomal
marker-trait associations were detected. Our findings illustrate minor evidence that correlation between UTT and FL is
partially determined by one or more sex-chromosomal QTL.
Introduction
Phenotypic correlation among traits arises from environmental and genetic covariance (Roff 1997; Lynch and Walsh
1998), the latter of which has consequences for selection for
genetically correlated traits (Lande and Arnold 1983; Lynch
and Walsh 1998). Genetic correlation is considered to be
caused by effects of single genes on multiple traits (pleiotropy) and/or multiple genes for different traits mapping to
the same chromosomal region (linkage disequilibrium)
(Lynch and Walsh 1998). Estimates of genetic correlation
between morphological traits (i.e., length and width) tends to
be the highest of all traits combinations (including
physiological, behavioral, and life-history traits; see Roff
1997), possibly because of the necessity for the coordinated
development of functionally and/or developmentally related
sets of morphological characters (‘‘morphological integration’’; see Olson and Miller 1958; Moreno 1994; Cheverud
1996). Morphological integration suggests that genes
affecting single traits or suites of characters should be
inherited as associated units (Cheverud 1996; Wagner 1996),
for which some quantitative trait locus (QTL) studies do
reveal empirical evidence (Leamy et al. 1999; Borowsky and
Wilkens 2002; Cai and Morishima 2002; Conner 2002).
There is also evidence for linkage and/or pleiotropy of
genetic factors affecting morphological, physiological, and
life-history characters (Knight et al. 2001; Cai and Morishima
2002), and it seems likely (given developmental and biochemical complexity) that cofunction of single genes for
different groups of traits (i.e., morphological and physiological) is occurring at some level (Wright 1968).
Phenotypic correlations of upper thermal tolerance
(UTT) with body length (fork length [FL]) and condition
factor (K, residual of logFW regressed on logFL) have been
observed in rainbow trout (Oncorhynchus mykiss) (Jackson et al.
1998; Perry et al. 2001), and nonzero genetic correlation
among these traits has been detected (Perry et al. unpub).
The partial genic basis for thermotolerance has been
demonstrated in this species with the detection of major
97
Journal of Heredity 2005:96(2)
Table 1. Significant associations between variations at two microsatellite loci and either fork length (FL) or condition factor (K) in
backcross families (Fam) of rainbow trout, where ‘‘n’’ ¼ number of individuals within full-sib family, Expt*[locus] represents experiment
day-[locus] interaction in within-backcross family trait modeling. Allele 184 at OmyFGT19TUF is in phase with the Y chromosome
(Danzmann, unpub.). Least-squares means (l) and standard error (SE) are given by allele. The proportion of variance associated with
a model term was calculated as r2 (SSterm/SStotal). Only results significant at a0.05 comparison-wise permutational thresholds are given
here (a priori significance: p,0.05*; p,0.01**; p,0.001***); bolded terms are significant at the a0.05 experiment-wise permutational
threshold (Few¼8.84) (Churchill and Doerge 1994). The result in BC Fam 25 for the association between segregation at
OmyFGT19TUF from sire 91–1 and K was marginally significant using permutational analysis (Few, a¼0.10¼6.89).
Effect
Allele
l6SE
F
p
r2
99
OmyFGT19TUF
0.0004***
0.0970
88
OmyFGT19TUF
4.37
0.0399*
0.0451
Dam
FL
99
OmyFGT19TUF
9.91
0.0022**
0.0831
Sire
K
88
OmyFGT19TUF
6.92
0.0102*
0.0749
Dam
K
103
Ssa20.19NUIG
173610.9
122611.3
89.1610.1
54.7612.1
17762.26
16762.06
0.027660.00783
0.0045660.00939
0.00070360.00758
0.023860.00733
13.8
UTT
184
210
184
210
190
210
184
210
75
87
4.80
0.0310*
0.0445
Fam
Sex
Trait
22
Sire
UTT
25
Sire
22
25
n
UTT QTL on several autosomal linkage groups (B, D, and S
[currently designated as RT-21, RT-14, and RT-6, respectively]; see Jackson et al. 1998; Danzmann et al. 1999;
Sakamoto et al. 2000; Perry et al. 2001). The new linkage
group designations follow Nichols et al. (2003). Body size
QTL have been detected on the linkage groups RT-6 and
RT-21 (Martyniuk et al. 2003; O’Malley et al. 2003), and there
is also evidence of epistatic effects on FL and UTT due to
interaction among genes on the linkage groups RT-21 and
RT-1 (the sex chromosomes) (Perry et al. 2003). If
correlation among these traits is partially controlled by
pleiotropy or linkage among multiple genes with different
effects, marker-trait associations for other traits should be
detected at regions containing UTT QTL, including the sex
chromosomes.
We tested the hypothesis that phenotypic correlations
between body size and UTT in rainbow trout might be
explained by pleiotropic effects of single genes or multiple QTL
for different traits mapping to the same chromosomal region.
We used three microsatellite loci located on the linkage
groups RT-6 (Ssa20.19NUIG), RT-21 (Omy325UoG), RT-14
(Ssa14DU ) (Jackson et al. 1998; Danzmann et al. 1999; Perry
et al. 2001) and a sex-linked microsatellite marker (OmyFGT19TUF) located near the sex-determining region on RT-1
(formerly linkage group 18; see Sakamoto et al. 2000) to detect
associations between putative QTL for FL, K, and UTT.
Materials and Methods
Backcross Half-sib Families
The thermotolerance selected family lines were initially
derived from a large (n¼2433) mixed population of rainbow
trout created from a sample of wild fish from the Ganaraska
River (Ontario, Canada), a commercial hatchery strain
(Goosens Trout Farm Ltd.), and an Ontario Ministry of
Natural Resources domestic strain (Maple, Ontario). Two
lines of rainbow trout with reciprocal upper thermal tolerance
(high (H) and low (L)) were created from this population by
98
divergent selection for resistance to acute thermal challenge
for three generations (Ihssen 1986). A two-year-old F1 hybrid
(HxL) male (91–1) between the two lines was bred to two H
females (88–5, 88–30) and one L dam (88–1) in 1993 to create
three backcross (BC) families (HxF1: 88–5391–1¼family 22;
88–30391–1¼family 41; LxF1: 88–1391–1¼family 25) (see
Table 1; Jackson et al. 1998).
Outbred Half-sib Families
Two first-generation (G0) males (‘‘grandsires’’) from two
commercial rainbow trout strains (Spring Valley (SVM1 and
SVM2) and Rainbow Springs (RSM1 and RSM2)) were bred
to 12 and 13 females (‘‘granddams’’) within each strain,
respectively, producing 35 purestrain second-generation (G1;
‘‘parental’’) families after mortality (see Table 2). The G1 fish
were interbred to create ten 232 diallel lots (40 full-sib
families) of purestrain and hybrid third-generation (G2)
progeny. The G2 families were reared at the University of
Guelph in sequential raceway sections or adjacent 7.8 L
basins or in 0.3 m circular tanks at the Alma Aquaculture
Research Station (Alma, Ontario).
Phenotypic Data Collection
Backcross and outbred fish were reared until 15–17 months
and 8–10 months postfertilization, respectively, and subjected to acute thermal challenge. During thermal challenge,
the water temperature was raised over one h from 108C to
25.78C (the critical thermal maximum (CTmax) for rainbow
trout reared at 10.08C (Currie et al. 1998)). Oxygen saturation
was above 80%. The tolerance time (min) at 25.78C until loss
of equilibrium (the inability of individual fish to maintain
normal orientation)—the effective time (ET) of thermal
tolerance (Fry 1971)—was considered to be the best
representation of UTT for the backcross and outbred
groups and is used throughout. Fork length (FL; length from
the snout to the fork of the caudal fin in millimeters; mm)
and fresh weight (FW; g) were also recorded for each fish.
Perry et al. Sex-linked Quantitative Trait Loci
Table 2. Significant associations between variance in fork length (FL), condition factor (K), and upper thermal tolerance (UTT)
and microsatellite loci linked to UTT QTL in rainbow trout. Loci with significant marker-trait associations are listed under each
common G1 parent, with the allele inherited from the G0 grandsire by the G1 parent indicated for the locus (given as size in base pairs).
Y-linked alleles at OmyFGT19TUF are underlined. Effect of allelic substitution (Lynch and Walsh 1998) given for each locus as lA 6
standard error. Significance of marker-trait associations are here given according to comparison-wise permutational estimates (Churchill
and Doerge 1994) (P,0.05*; P,0.01**); results significant (P,0.05*) at experiment-wise thresholds are given in bold. UTT marker
results are reported only for the sex-linked marker OmyFGT19TUF; autosomal UTT QTL are described in Jackson et al. (1998),
Danzmann et al. (1999), and Perry et al. (2001, 2003). Proportion of variance explained by putative QTL (r2) was determined as ratio of
SSlocus/SStotal. Significant effects of allelic substitution at the locus within each half-sib family are indicated under the column lA 6 SE
in cases where marker-full sib-family interaction was observed (P,0.10y; P,0.05*; P,0.01**; t-test (PROC GLM, SAS 1998).
G0SVM2
SVM1
G1 parent (Sex)
Allele, trait
SV-2–2 (F)
Ssa20.19NUIG2-79,FL
lA 6 SE
3.60 6 1.27*
2.77 6 1.56
P
r2
G1 parent (Sex)
Allele, trait
lA 6 SE
P
r2
0.0245*
2.49
SV-21–1 (F)
Ssa14DU1-133,K
0.0306 6 0.0103
0.0265*
0.0346*
3.48
3.15
Ssa14DU2-133,K
0.0598 6 0.0154*
0.00150 6 0.0137
SV-5–6 (F)
Omy325UoG1-113,FL
8.05 6 2.38
0.0125*
6.78
SV-24–10 (F)
Ssa14DU1, 3, 4–123,FL
8.82 6 2.88
0.0398*
7.68
SV-6–7 (M)
OmyFGT19TUF1-185,FL
6.84 6 1.73
0.0065**
6.42
SV-30–5 (F)
OmyFGT19TUF1-193,FL
6.93 6 2.19
0.0281*
5.17
0.0429*
0.0122*
4.56
6.52
SV-32–1 (M)
Omy325UoG2-113,K
Ssa20.19NUIG2-87,K
6
6
6
6
0.0289*
0.0290*
2.69
2.63
0.0141*
0.0457*
3.11
1.77
SV-8–2 (F)
OmyFGT19TUF1-197,FL
OmyFGT19TUF1-197,K
SV-10–6 (M)
Omy325UoG2-113,FL
OmyFGT19TUF2-185,UTT
6.48 6 2.22
0.0428 6 0.0117
5.01
3.81
3.89
11.8
6
6
6
6
1.78y
1.68
3.91
3.37
RSM1
G1 parent (Sex)
Allele, trait
RS-1–2 (F)
Ssa14DU2-137,K
RS-1–9 (M)
OmyFGT19TUF2-165, K
lA 6 SE
P
r2
G1 parent (Sex)
Allele, trait
RS-25–8 (F)
0.00696 6 0.0143
0.0419* 2.36 OmyFGT19TUF1-185,K
0.0392 6 0.0102**
0.059 6 0.018*
0.019 6 0.017
RS-28–5 (M)
0.0290* 3.03 Ssa20.19NUIG2-83,FL
OmyFGT19TUF1-167, FL
0.05 6 0.01
3.78 6 1.05
RS-31–10 (M)
0.0180* 9.55 OmyFGT19TUF1-167, K
0.0135* 6.23
RS-9–7 (M)
OmyFGT19TUF1-165,UTT
6.41 6 1.73
0.0098* 3.43
RS-15–6 (M)
Ssa14DU1-137,FL
RS-18–2 (F)
Omy325UoG2-115,FL
Ssa14DU1-141,K
0.0234
0.0213y
0.0224
0.0206
RSM2
RS-5–10 (M)
Ssa20.19NUIG1, 3, 4–83,K
OmyFGT19TUF1-165, FL
RS-12–9 (F)
Ssa20.19NUIG1-83,K
0.0416
0.0508
0.0463
0.0438
0.0333 6 0.00935
RS-34–7 (F)
0.0123* 3.85 OmyFGT19TUF2-185, FL
4.50 6 1.40
0.0264* 4.67
3.02 6 1.22
2.93 6 1.38
0.0299 6 0.01
0.0347* 2.39
0.0305* 2.41
0.0159* 3.01
lA 6 SE
P
0.0358 6 0.0122 0.0412*
r2
4.58
1.08 6 0.306* 0.0109*
6.67
0.52 6 0.340 0.0019** 9.56
9.16 6 2.02
0.0275 6 0.0084 0.0226*
2.84
5.92 6 2.38y
4.99 6 2.89
2.96
0.0416*
99
Journal of Heredity 2005:96(2)
Table 2.
Continued
RSM1
G1 parent (Sex)
Allele, trait
Ssa14DU2-141,K
RS-21–6 (M)
OmyFGT19TUF1-165, K
RS-21–9 (F)
Omy325UoG2-137,K
RSM2
lA 6 SE
P
r2
G1 parent (Sex)
Allele, trait
lA 6 SE
P
r2
0.0348*
4.72
RS-37–10 (M)
OmyFGT19TUF1-167,UTT
9.82 6 2.76
0.0169*
2.85
0.0017**
5.25
0.00346 6 0.0137
0.0632 6 0.0149**
0.0355 6 0.0118
0.0310 6 0.0122y
0.0426 6 0.0115**
1
Main effects marker-trait association.
2
Interactive marker-full sib-family association with phenotype.
3
One of the noncommon parents crossed to this individual was an identical heterozygote (on homozygote G2 used in testing in that half-sib family).
Indicates inability to unambiguously identify G0 allele due to G0 homozygosity or identical heterozygosity between grandsire and granddam.
4
Condition factor (K) was calculated as the residual of the
regression of log(FW) on log(FL) (log(g)log(mm)1)
(Barton 1996) within lot. Previous work has shown that
physical traits such as K and FL affect thermal tolerance
(Jackson et al. 1998; Perry et al. 2001; Perry et al. 2003), and
these were therefore used as covariates in tests for UTT QTL
(PROC REG; SAS 1998). FW was highly collinear (variance
inflation factor 10.0; SAS 1998) with FL in the half-sib
progeny of each of the G1 parents; thus, only FL and K were
tested as possible covariates for UTT. In raceway-reared
outbred G2 families, both K (P,.001***) and UTT
(P,.0001****) were affected by the location of their family
relative to the raceway inflow during early rearing, and trait
values were therefore adjusted for this effect in these groups.
No raceway positional effects were seen on FL (P¼0.2716).
Molecular Methodology
DNA extraction was performed using a modified phenolchloroform protocol (see Bardakci and Skibinski 1994) or
a commercially available complete genomic extraction kit
(IsoQuick Nuclear DNA Extraction Kit, ORCA Research).
The determination of genotypes at the four microsatellite loci
(Omy325UoG, Ssa14DU, Ssa20.19NUIG and OmyFGT19TUF) was conducted by using radioactively labeled primer
or direct incorporation of Tamra-labeled dCTP to visualize
fragments. PCR procedure consisted of an 11 ll reaction
volume containing 30 ng template DNA, 20 mM Tris-HCl
(pH 8.4), 50 mM KCl, and 0.25 units of Taq DNA polymerase.
The radioactive procedure employed 0.17 pmol g-P33 endlabeled primer, 5 pmol unlabeled primer, and 0.175 mM of
each dNTP. The forward primer was radioactively labeled for
Omy325UoG and the reverse primers for Ssa20.19NUIG,
Ssa14DU and OmyFGT19TUF. Fluorescent amplification
employed 5 pmol each primer (unlabeled), 4 mM Tamralabeled dCTP, and 0.375 mM dNTPs. Amplified fragments
were run on 6% polyacrylamide gels with a known length
standard (M13 sequence ladder or Tamra 350 bp ladder) for
1500–1700 V for 1–2 hours and were visualized by
autoradiographing dried acrylamide gels on Kodak Biomax
100
MR film (radioactive procedure; Jackson et al. 1998) or by
scanning in a Hitachi FMBIOä imaging system (fluorescent
procedure). The probable sex of G2 individuals was scored
using OmyFGT19TUF genotype. The recombination rate
between OmyFGT19TUF and SEX is reported to be low in
some experimental families (,2%; Sakamoto et al. 2000),
although Nichols et al. (2003) reported higher recombination
rates between this marker and the sex locus in a doubled
haploid mapping family of rainbow trout. OmyFGT19TUFSEX recombination in the G1 generation was roughly
equivalent to that found in the experimental families of
Sakamoto et al. (2000) (data not shown).
Backcross Family QTL Detection
Since not all backcross families could be challenged during
a single experiment, thermal tolerance tests were done over
several experiment days (Jackson et al. 1998). As a result, the
fish from the different experiments varied by age. QTL
detection was therefore performed within each of the
backcross families to determine associations between traits
and segregation from sires and dams using the model
yijkl ¼ l þ ai þ /j þ ai /j þ )m¼bm Xm þ eijkl
ð1Þ
in PROC GLM (SAS 1998), where yijkl is the phenotype of
a backcross individual; ai represents the fixed effect of allele i
at one of the loci; /j represents the fixed effect of experiment
day j; ai/j is the interaction of allele i with experiment j; bmXm
represents partial regression coefficients for each of m
covariates correlated with UTT (from among K and FL)
after stepwise backward regression (a for removal¼0.05);
and eijkl was the residual error. Phenotype and genotype were
randomly permuted 1,000 times; and the same linear model
was rerun within each half-sib family for each permutation of
the data. The fiftieth highest F-value (fifth percentile) was
selected as the comparison-wise significance threshold
(Churchill and Doerge 1994). Since analyses were run
separately within each full-sib backcross family, experimentwise significance thresholds at the a0.05 level were selected by
Perry et al. Sex-linked Quantitative Trait Loci
permuting phenotype on genotype separately for all three
traits within each backcross family. Each possible test was
then rerun within each backcross full-sib family, and the
highest F-value was then recorded. A total of 1,000 such
permutes were run, and the fifth percentile of the set of
maximum F-values for all families was used as the
experiment-wise threshold (Churchill and Doerge 1994).
We estimated allelic substitution effects (a; Lynch and Walsh
1998) based on phenotypic differences between progenyinheriting alternate alleles (ai) from a single parent in the
half-sib model at one locus (ie., P (phenotype) ¼ lG þ ai
rather than P ¼ lG þ ai þ aj since the latter represents
a diploid model; Lynch and Walsh 1998).
Outbred Family QTL Detection—G1 Parents
The association of alleles segregating from G1 parents with
trait phenotype in the G2 half-sib families was determined in
the general linear model (PROC GLM; SAS 1998)
yijkl ¼ l þ ai þ /j þ ai /j þ )m¼bm Xm þ eijkl
ð2Þ
where yijkl is the trait value for individual k from a G2 half-sib
family; ai represents the fixed effect of segregation at locus i
from the common G1 parent; /j is the fixed effect of
noncommon G1 parent j in the half-sib family; ai/j
represents the interaction of locus i in the common parent
with the genetic background of each noncommon parent in
each half-sib family; bmXm represent up to two partial
regression coefficients for covariates affecting UTT (K and/
or FL) not rejected by stepwise backward regression; and eijkl
is the random residual. The interaction of putative QTL with
full-sib family was included in order to account for effects of
genetic background on QTL expression. QTL tests were
performed across both half-sib families of each common
parent (rather than within each full sib-family, as above)
because all fish within single diallel lots were tested on the
same day (i.e., the design was completely cross-classified) and
in order to limit the number of independent tests performed.
Comparison-wise and experiment-wise, significance thresholds (Churchill and Doerge 1994) were assessed using permutation as described above, with maximum F-values
calculated within half-sib family and summed over all halfsib families in accordance with the within-family analysis.
Outbred Family QTL Detection—Grandsire Analysis
The outbred pedigree and the genotypic data were used to
determine the origin and inheritance of alleles at each locus
as well as their association with the traits across the complete
set of grandprogeny of each of the outbred grandsires
(SVM1, SVM2, RSM1, and RSM2) in the model
yijgk ¼ l þ ai þ cj ðai Þ þ ug ðcj Þ þ )m¼1 bm Xm þ eijgk ð3Þ
where yijgk is the phenotype of G2 grandprogeny k of the
grandsire; ai is the effect of allele i from the grandsire; cj(ai) is
the nested effect of G1 half-sib family parents (cj) (i.e.,
progeny of the grandsire) by allele received from the latter (ai);
ug(cj) is the effect of the G1 full-sib family parents (ug) nested
within the half-sib family G1 parent (i.e., full-sib familyspecific effects); bmXm is the partial regression coefficient for
traits correlated with UTT; and eijgk is the random residual.
Nesting of effects was used rather than simple interaction
because G1 individuals were not replicated over G2 diallel lots
(i.e., an incompletely cross-classified model). Marker-trait
associations for UTT were only tested using the sex-linked
marker OmyFGT19TUF because autosomal results for these
populations have already been reported (Jackson et al. 1998;
Danzmann et al. 1999; Perry et al. 2001). This model
permitted tests of main effects of putative QTL and of effects
conditional on genomic background. Trait value by grandsire
allele was determined using means calculated with the
MEANS statement in PROC GLM (SAS 1998). ShapiroWilk tests (Steel and Torrie 1980) indicated that model
residuals deviated from normality across the combined set of
grandprogeny from each grandsire, and so optimal transformations of trait values were determined in each grandsire
family using a BoxCox macro (BoxGLM, M. Friendly, York
University) in SAS (1998) prior to analysis. Where this was
unsuccessful at finding a suitable transformation, data was
standardized (r2¼1, l¼0) prior to analysis.
Effects of Strain and Sex
We also estimated overall effects of sex on FL, K, and UTT to
determine whether males or females exhibited phenotypic
differences suggestive of simple sex-limited expression (i.e.,
uniformly greater or lower trait values in males compared to
females) rather than sex-linked genes. Sex of G2 individuals was
inferred using the OmyFGT19TUF genotype and the pedigree
as described above. The effects of genetic sex on these traits
were determined separately for each trait in SAS (1998) for the
entire pedigree population using the general linear model
yijkl ¼ l þ ai þ /j þ ai ðcg Þ þ cg ð/j Þ þ eijkl
ð4Þ
where yijgk is the phenotype of G2 individual k; ai is the effect
of genotypic sex; /j is the effect of strain background (SV, RS,
or hybrid); ai(cg) is the nesting of genotypic sex within the
progeny of G1 sire g; cg(/j) is the effect of G1 sire g nested
within strain j; and eijkl is the random residual. G1 sires were
included as nested effects because they were not replicated
across all G2 lots (see above). Main and nested effects of sex
were included to account for effects of sex-linked loci across
the pedigree of descendants from a single grandsire (i.e.,
additive genetic variance) and for effects within specific fullsib grandprogeny families (i.e., nonadditive genetic and
environmental effects). All three traits were standardized
(l¼0, r2¼1) before analysis in order to normalize data.
Results
QTL Analysis in Backcross Families
The significance of potential covariates for UTT QTL tests at
OmyFGT19TUF varied by family. FL had a strong and
101
Journal of Heredity 2005:96(2)
negative correlation with UTT in BC family 22 (F1,120¼9.78;
P¼0.0020**; b¼0.94960.303 mm) and family 41
(F1,87¼249; p¼0.0001; b¼2.1860.138 mm). Condition
factor was not correlated with UTT in any lot (P..05). No
significant covariates with UTT were detected in family 25
(P..05). Mean UTT phenotype was 15267.3 min in BC
family 22, 72.466.55 min in family 25, and 211610.0 min in
family 41, or 13863.78 min overall. The UTT variance within
each BC family ranged from 4583 to 15443 min2 (BC families
25 and 41, respectively). Both FL and K were frequently
associated with thermal tolerance in the outbred families; but
in contrast to the findings from the backcross families,
positive correlations were also observed. Correlations among
traits ranged from 0.371 to 0.350 for UTT-FL and from
0.210 to 0.345 for UTT-K (data not shown).
Additive effects of UTT QTL on autosomal linkage
groups RT-21, RT-14, and RT-6 in the backcross and
outbred families have been previously reported elsewhere
(Jackson et al. 1998; Danzmann et al. 1999; Perry et al. 2001)
and are not included here. Further, we only describe results
that were significant (P,.05*) at comparison-wise permutational thresholds or lower in this paper. Segregation at the
sex-linked marker OmyFGT19TUF from the F1 sire 91–1 was
significantly associated with UTT at the experiment-wise
threshold (Few¼8.84) in family 22, explaining a major
proportion (9.7%) of phenotypic variance (Table 1).
Segregation at this marker from the same sire was also
associated with UTT in family 25 but was responsible for
a smaller proportion (r2,5%) of variance and thus was
significant only at the comparison-wise threshold (Table 1).
Allele 184 from this sire (the Y-linked allele) was associated
with high thermal tolerance in both of the above BC families.
There was marginal a priori evidence that this association
was reversed in BC family 41 (P¼0.0962; r2¼0.0284;
l184¼259 6 15.3, l210¼296 6 15.1), but this association
was not significant at the permutational threshold. Progeny
of 91–1 in family 25 inheriting the Y-linked allele (184) at
OmyFGT19TUF had a K 0.32 log(mm)log(g)1 greater than
that of their female sibs (Table 1), but this was only
marginally significant (F1, 87¼6.92; Few, a¼0.10¼6.89).
Segregation at OmyFGT19TUF from 91–1 controlled 7.5%
of phenotypic variance in K in this full-sib family. FL was
also significantly associated with segregation at OmyFGT19TUF from dam 88–5 in family 22 at the experiment-wise
threshold (P,.01; Table 1). Progeny inheriting the 190 allele
from dam 88–5 were approximately ten mm longer than their
sibs inheriting the 210 allele, with OmyFGT19TUF explaining
over 8% of phenotypic variance in FL (Table 1).
There was relatively little evidence of autosomal markertrait associations for FL and K (but see Jackson et al. 1998
and Danzmann et al. 1999 for UTT results). K was
significantly associated with segregation at Ssa20.19NUIG
from dam 88–5 at comparison-wise thresholds, with
full-sib individuals inheriting allele 87 being 0.0231
log(mm)log(g)1 larger than those inheriting allele 75 (Table
1). Experiment (i.e., age) did not have significant effects on K
overall but did affect FL significantly in the backcross
families (P,.05*). There was no evidence of GxE interaction
102
between putative QTL and experiment day within the
markers used here (P..05).
QTL Analysis in Outbred Families—Sex-linked Regions
We estimated the a0.05 threshold for significant additive
effects of single QTL in the outbred families as
Few1, a¼0.05¼7.72 and the threshold for the interaction of
specific putative QTL with full-sib family background as
Few2, a¼0.05¼7.84. Variation at the sex-linked locus,
OmyFGT19TUF, was frequently associated with variance in
phenotype (particularly for FL) in several of the G2 families
and primarily occurred via segregation from sires (Table 2).
However, there was variability in the direction of the
relationship between phenotype and sex; the size and thermal
tolerance of G2 males relative to females was dependent on
the specific origin of the Y-linked allele. By example, male
progeny of sire SV-6–7 were significantly longer (at the
experiment-wise threshold) than female half sibs by an
average of 6.8 mm (Table 2). However, male progeny of RS G1
sires RS-28–5 and RS-5–10 (inheriting the Y-linked allele
from RSM2 and RSM1, respectively) were on average 9.2 and
3.8 mm shorter than their half-sib sisters (Table 2), although
the results from the latter sire were only marginally (P,.10)
significant at the experiment-wise threshold. FL was also
associated with OmyFGT19TUF in the half-sib progeny of
dams SV-8–2, SV-30–5, and RS-34–7 using comparison-wise
thresholds (Table 2). The proportion of variation in FL
accounted for by OmyFGT19TUF in the outbred families
ranged from 3.0–9.6% of total phenotypic variance.
OmyFGT19TUF was marginally associated with UTT at the
experiment-wise threshold in the half-sib progeny of G1 sire
RS-9–7, where average male UTT was 6.4 6 1.7 min lower
than that of their female half sibs (Table 2). However, male
half-sib progeny of G1 sire RS-37–10 were 9.8 6 2.9 min
more thermotolerant than their half-sib sisters (Table 2).
Additionally, there was evidence of interaction between allelic
inheritance at OmyFGT19TUF from SV-10–6 and the dams of
the half-sib families at the comparison-wise threshold
(although no main effects were observed) (Table 2).
Genetic variation at OmyFGT19TUF was also associated
with phenotypic variance in K in the outbred families.
However, where such associations were significant, males
were always heavier for their length (where such differences
were significant) than female half sibs, rather than
varying between being longer or shorter than females. Male
progeny of three Rainbow Springs G1 sires (RS-1–9, RS21–6 and RS-31–10) were significantly heavier (0.01–0.04
log(mm)log(g)1) for their length than their female counterparts (Table 2). Genotype at OmyFGT19TUF was also
associated with K in the progeny of two dams, SV-8–2 and
RS-25–8, with the former association being marginally
significant at the experiment-wise threshold (P,.10; Table 2).
QTL Analysis in Outbred Families—Autosomal Regions
Interaction between genotype at Omy325UoG and full-sib
family was significantly associated with variation in K in the
Perry et al. Sex-linked Quantitative Trait Loci
Table 3. Association of segregation at the sex-linked microsatellite OmyFGT19TUF with fork length (FL) and upper thermal
tolerance (UTT) in outbred (G2) rainbow trout using a grandsire QTL inheritance model. Degrees of freedom (df), number of
grandprogeny per test (N), offspring sex, mean-trait value by offspring sex, probability for rejection of the null hypothesis (P), and
proportion of variance controlled by each factor (r2) are given for each individual test by grandsire. Sex was determined in the G2
families using OmyFGT19TUF genotype and the three generations of the population pedigree. For marker-FL associations in the
descendants of SVM1 and RSM2, data was normalized using transformations derived with BoxGLM (M. Friendly, York University), while
UTT data was standardized (l, r2) prior to analysis. FL and UTT means were calculated using the MEANS statement in PROC
GLM (SAS 1998) and are given in mm and min retransformed to their original values, respectively. Significance of effects given as
P,0.10y, P,0.05*; P,0.01**, P,0.001***; P,0.0001****. Proportion of variance explained by each term (r2) determined via REML
(PROC VARCOMP; SAS 1998).
Source of variation
Trait
SVM1
OmyFGT19TUF
FL
G1 sire(OmyFGT19TUF)
Fam(G1 sire)
RSM2
OmyFGT19TUF
G1 parent(OmyFGT19TUF)
Fam(G1 parent)
OmyFGT19TUF
G1 parent(OmyFGT19TUF)
Fam(G1 parent)
df
2
N
Sex
l (SD)
344
262
#
$
20.7 (1.55)
18.3 (1.23)
8
10
UTT
2
FL
7
9
2
#
$
291
280
#
$
48.3 (36.0)
61.7 (36.3)
11.2 (3.78)
10.5 (3.39)
7
9
progeny of dam RS-21–9 (Table 2) using the experiment-wise
threshold (Few1, a¼0.05¼7.72), suggesting interaction between
putative QTL and genetic factors from the noncommon
parents (ai/j in Equation 2) or effects associated with
environmental variance undetected in our analyses. However,
no other significant results for autosomal markers were
detected, although three tests indicated marker-trait associations for autosomal linkage groups marginal (P,.10) at Few:
FL was associated with Omy325UoG in the half-sib families of
SV-5–6; Ssa20.19NUIG with K in RS-12–9’s progeny; and
interaction of Ssa20.19NUIG with full-sib family was
associated with variance in FL in the families of RS-28–5
(Table 2). Effects of allelic substitution at autosomal loci were
between 4–8 mm for FL and between 0.02–0.05 log(gmm1)
for K and accounted for up to 9.5% of phenotypic variance.
Where effects of putative QTL linked to autosomal markers
varied by full-sib family, phenotypic variance explained by
these effects ranged from 2.4–5.3% but were usually less than
3.5% (Table 2).
QTL Analysis in Outbred Families—Grandsire Model
G2 fish receiving different alleles at OmyFGT19TUF from
RSM2 had significantly different UTT (P,.05). Segregation
at this marker accounted for 0.36% of variance in their UTT
overall (Table 3). Male G2 grandprogeny of RSM2 (inheriting
the Y-linked 167 allele) from that grandsire had a UTT
significantly lower than his granddaughters (X-linked
allele¼185). Similarly, significant effects of OmyFGT19TUF
on FL were observed in the grandprogeny of SVM1 and
RSM2 (Table 3). Male G2 grandprogeny of SVM1 (inheriting
the Y-linked allele 185 at OmyFGT19TUF) were 2.3 cm
r2
P
0.0188*
0.00334
0.0803y
,0.0001****
0.00471
0.624
0.0155*
0.00358
0.9828
,0.0001****
0.0085**
0.000635
0.00549
0.1397
,0.0001****
0.00632
0.0288
longer than his female grandprogeny, with inheritance at
OmyFGT19TUF accounting for 0.33% of variation in FL. In
contrast, male grandprogeny of RSM2 were 4.3 cm smaller
than their female siblings, and locus effects accounted for
0.63% of variation in FL overall. No effects of autosomal
loci on FL or K were detected, and K was not significantly
associated with segregation at the microsatellite loci from any
of the grandsires. Effects of full-sib families and G1 parents
on FL and K were highly significant in almost all grandsire
tests (Table 3). The proportion of variance explained by all
factors used in the grandsire model, including putative QTL
effects, was considerably lower than estimates of similar
effects in single-generational modeling (,5%), despite the
estimates of significance associated with them. This effect
has been reported elsewhere (Perry et al. 2001) and likely
represents error variance introduced by a mixture of complex
environmental and genetic effects in the multigenerational
analysis. There was some residual heterogeneity even after
data transformation when analyzing OmyFGT19TUF-UTT
associations in the grandprogeny of RSM2 using the ShapiroWilk test (P,.01**), but this test may be overly sensitive
(Steel and Torrie 1980). Normality plots appeared correct on
inspection (not shown), and tests using standardized data
(l¼0, r2¼1) supported these results.
Sex and Strain Effects
Male G2 fish were significantly heavier for their length
overall than female G2 fish (term ai in Equation 4; Table 4),
but the effects of sex on FL varied by the G1 sire of origin
(term ai (ck) in Equation 4). Fish from the two strains
differed significantly in FL and K (Table 4; Equation 4).
103
Journal of Heredity 2005:96(2)
Table 4. Effects of sex and strain on fork length (FL) and condition factor (K) in a pedigree of outbred rainbow trout (O. mykiss).
Number of observations (N), means (l), and standard deviation (SD) of standardized FL (l¼0, s2¼1) and K given by sex. Differences
in N among traits indicates unavailability of some data. Means marked by superscript letters (i.e., a) are significantly different at a¼0.05.
The significance of effects in the models is given as P,0.10y, P,0.05*, P,0.01**, P,0.001***, P,0.0001****. Proportion of variance
explained by each term (r2) was determined using PROC VARCOMP (SAS 1998).
Source
UTT
Sex
Strain
Effect
N
l 6 SD
M
F
SV
RS
HYB
1268
1291
556
885
1118
0.0157
0.0316
0.257
0.138
0.000439
6
6
6
6
6
1.01
0.980
0.832
0.889
1.12
Sire (strain)
Sex (sire)
FL
Sex
Strain
M
F
SV
RS
HYB
1269
1293
559
885
1118
0.0209
0.0202
0.674
0.342
0.0192
6
6
6
6
6
1.01
0.979
1.11
0.768
0.930
Sire (strain)
Sex (sire)
K
Sex
Strain
M
F
SV
RS
HYB
1267
1288
553
885
1117
0.028
0.0334
0.161
0.00889
0.0785
Sire (strain)
Sex (sire)
Hybrid and purestrain SV fish were significantly longer
(0.246 mm and 0.210 mm, std., respectively) than RS
purestrain fish, but they were not significantly different from
each other. Hybrids were significantly thinner than either SV
or RS fish (0.207 and 0.104 log(gmm1), respectively), while
K was not significantly different for SV and RS fish. G1 sires
also had strong effects on the FL and K of their progeny.
Genetic sex accounted for less than 1% in the variation in FL
and K across the entire pedigree. UTT was not significantly
associated with sex per se in this analysis (P..05), but there
were large differences between the strains, with RS having
a higher UTT (Table 4).
Discussion
Sex-Linked Genic Effects
Using single-generational and grandsire modeling, we
detected evidence that FL and UTT were associated with
sex in juvenile rainbow trout via segregation at a sex-linked
microsatellite marker (OmyFGT19TUF). However, the
phenotype of males relative to their female sibs varied by
the origin of the Y chromosome. For example, male G2
grandprogeny of RSM2 inheriting the Y-linked allele at
OmyFGT19TUF from him were smaller (but more thermotolerant) than their female siblings, while male G2 fish
inheriting their Y chromosome from SVM1 were larger on
average than female half sibs. Similarly, over the entire
104
r2
P
6
6
6
6
6
0.963
1.03
1.17
0.970
0.914
0.5417
6.563105
,0.0001****
0.0295
,0.0001****
0.1864
0.0271
4.283103
0.2908
2.223104
,0.0001****
0.00499
,0.0001****
0.0471*
0.0129
0.00607
0.0304*
0.00163
0.0199*
0.00273
,0.0001****
0.7846
0.00751
0.00487
population, effects of sex on FL were significant but variable
by the G1 sire (the individual responsible for determining sex
in their progeny). Therefore, we believe that our evidence
suggests the existence of one or more sex-linked QTL with
moderate effects (3–10% of variance) on FL and possibly
UTT. In conjunction with findings by Perry et al. (2003) of
epistasis involving sex-linked genes, this explanation seems
more reasonable than simple sex-limited trait expression
because male phenotype relative to that of female sibs varied
by strain rather than being uniform. Previous work also
indicates sex-linked genes associated with growth in
salmonids (Allendorf et al. 1994; Forbes et al. 1994; Perry
et al. 2003) and in other taxa (Rohrer and Keele 1998a,b;
Davis et al. 2001). However, the extensive linkage-group
rearrangements associated with the salmonid sex chromosomes (Woram et al. 2003) make it difficult to ascertain
homologies for specific marker regions. If sex-linked QTL
are affecting these traits, lower recombination rates over
centromeric regions in male salmonids compared to females
(see Sakamoto et al. 2000) should preserve marker-QTL
linkage and result in a greater rate of QTL detection by
measuring marker segregation from sires to progeny, which
was in fact observed at OmyFGT19TUF (12 of 17 total
results significant a priori). However, we acknowledge that
effects of sex itself may have partially confounded our results
and that differences in the nature of sex limitation between
populations, if present, might also have caused differences
in relative male/female trait value between RS and SV fish.
Perry et al. Sex-linked Quantitative Trait Loci
This may be a topic for future investigation. In contrast to
the above results, males did have higher K than females
across the entire population, irrespective of sire and/or
Y-chromosomal origin. This does suggest sex limitation of
condition factor in juvenile male salmonids (see also Sylvén
and Elvingson 1992; Elvingson and Johansen 1993),
although such sex limitation would be necessarily partial
since estimates of additive heritability for all three traits are
high (h2a.0.4) in this population (Perry et al. unpub). We
also note that the two groups of fish used in this study (the
outbred and BC families) were not of identical age at the time
of experimentation (8–10 months and 15–17 months
postfertilization, respectively). Ontogenetic variation in
gene activity, previously observed in this species (Martyniuk
et al. 2003), may have partially biased our results. Unfortunately, we cannot differentiate between effects on QTL
detection arising from ontogeny and the genetic background
of the lineages themselves (since selective histories necessarily varied between the two; see Jackson et al. 1998; Perry
et al. 2001), although results from the BC groups tend to
support those of the outbred families.
Although marker-trait associations at the sex chromosomes in mammalian species are usually assumed to reflect
exclusive X-linkage of functional alleles (Rohrer and Keele
1998a,b; Davis et al. 2001), because the typically degenerate
Y chromosome (compared to the X chromosome) appears
to carry only genes for reproductive traits (see Capel 2000;
Charlesworth 2001; Jobling et al. 2001), the salmonid sex
chromosomes are believed to be highly homologous (i.e.,
nondegenerate Y chromosome) (Thorgaard 1983; Hartley
1987; Colihueque et al. 2001). In conjunction with our results
for variance in marker-trait associations at OmyFGT19TUF
by sire origin and the findings of Perry et al. (2003), we thus
also consider the possibility that the salmonid Y chromosome may carry functional alleles at sex-linked genes for
nonreproductive traits (i.e., FL and UTT).
Selective effects of Y-linked alleles at sex chromosomal
QTL for size and/or thermoresistance might result in
fitness differences between the sexes and sex ratio changes
unrelated to the processes of sexual differentiation and
reproduction (see Rikardsen et al. 1997; Spidle et al. 1998;
Rhodes and Quinn 1998; Johnsson et al. 1999). However,
our evidence for polymorphisms of Y-linked QTL alleles
suggests that such changes would occur within specific sire
lines (i.e., for specific Y chromosomes) and might thus be
invisible at the populational level because of the existence
of other polymorphisms. Long-term intrapopulational
changes in Y-chromosomal diversity could be one indicator
of such a phenomenon. Additionally, fork length itself may
not be an entirely neutral trait in this regard, since it may be
under some degree of sexual selection. Polymorphisms at
functional Y-linked QTL alleles for growth might be
involved with the production of the major life-history
dimorphisms for size and reproductive characters extant in
male salmonids (see Gross et al. 1996) and likely partially
explain the high heritability exhibited for precocious
maturation (‘‘jacking’’) in salmonid fish (Hankin et al.
1993; Heath et al. 1994).
Autosomal Multi-trait Associations
There was relatively little evidence for effects of autosomal
QTL linked to Omy325UoG, Ssa14DU, and Ssa20.19NUIG on
the two growth traits (FL and K) in these populations. We
may thus be able to reject the hypothesis that genetic and
phenotypic correlations among UTT and the two growthrelated traits in this population are due to linkage among
different QTL variants or pleiotropy (where it applies to the
autosomal regions surveyed here). Autosomal QTL for
growth/size characters have been detected in various
genomic regions (Robison et al. 2001; Martyniuk et al.
2003), including those used here (O’Malley et al. 2003), and
evidence of pleiotropic/linked genes for traits in different
categories has also been detected by genetic analysis in
salmonid fish (Allendorf et al. 1983; Ferguson and Danzmann
1985). Martyniuk et al. (2003) indicated that allelic variation
on linkage group 21 accounted for up to 9% of body weight
variation in the progeny of SVM1 in the previous generation
of the same stock. Our failure to detect similar QTL effects
might have been the result of other genetic or environmental
effects, such as the confounding of minor autosomal QTL
effects by those of unassayed QTL elsewhere in the genome.
Most support for pleiotropic effects of single genes or
genomic regions occurs for functionally integrated morphological traits (Leamy et al. 1999; Vaughn et al. 1999; Wayne
et al. 2001; Cai and Morishima 2002; Borowsky and Wilkens
2002; Conner 2002). However, physiological complexity
leads to the expectation that pleiotropy should be commonly
observed, because single genes will frequently be involved
with multiple biochemical pathways (Wright 1968). Our
results, in conjunction with Perry et al. (2003), suggest that
the genetic control of traits in very different categories
(morphological, physiological; see Roff 1997) might be
partially nonindependent due to pleiotropy or linkage
disequilibrium. If QTL for UTT and FL with alleles of
variable effects on different traits are indeed coincident on
the sex chromosomes, this suggests that antagonistic
pleiotropy could occur via sex-chromosomal genes and
might partially explain negative phenotypic (Jackson et al.
1998) and genetic correlations (Perry et al. unpub) between
UTT and FL. Although the maintenance of genic polymorphism via antagonistic pleiotropy (including sex-limited
traits) is debatable (Curtsinger et al. 1994; Lynch and
Walsh 1998; Hedrick 1999), complexities in sex-limited
expression, coupled with sex-linkage of QTL on more than
two fitness-related traits, have not been modeled. The
eradication of Y-linked variants with antagonistic effects on
multiple nonreproductive characters (most plausible where
functional content of the Y chromosome is unreduced) might
be limited by the actual Y-chromosomal diversity for alleles at
sex-linked loci with nonantagonistic effects on trait values.
Acknowledgments
We thank the staff of the Alma Aquacultural Research Station and the Hagen
Aqualab for assistance with fish rearing and experimentation. J. P. Gibson, E.
G. Boulding, and J. A. Robinson helped with useful comments regarding
105
Journal of Heredity 2005:96(2)
statistics and presentation. H. Allen, L. Mancuso, C. Mandzuk, and R. Woram
helped with experimentation. This research was funded by NSERC.
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Received April 2, 2003
Accepted August 15, 2004
Corresponding Editor: Lisa Seeb
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