significant deviation from expected proportions is discussed with respect to linkage or selection.
2. Material and methods
2.1. Experimental protocol F individuals were obtained by crossing females of an experimental domesticated
1
Ž .
stock INRA with wild Mediterranean males from the Reverotte River, a tributary of
`
the Doubs River. The INRA strain was funded six generations ago from fish farm strains Ž
. of different origin and immediately closed
no introduction latter . The strain is propagated over generations, with a large effective size, 60 males and 60 females, per
Ž generation. The initial level of variation is high Krieg and Guyomard, 1985; Estoup et
. al., unpublished and according to the effective population size, very little increase of
inbreeding and relatedness is expected. Populations from the Doubs basin are Mediter- Ž
. Ž
. ranean trout, as shown by Presa et al. 1994 and Largiader et al. 1996 . Moreover,
genotyping of 50 F with several loci confirmed that all males used for the crosses
1
Ž .
belong to the Mediterranean form Guyomard, 1997 . No relatedness exists between domestic and Mediterranean trout.
Two types of crosses were performed using unrelated mates, the offspring of each Ž
. mate constituting a numbered set given below .
Ž .
- Two crosses involving a hybrid F female fH1 and fH2 and a domestic male
1
Ž .
mD1and mD2 . Hereafter, these crosses will be named BC1 and BC2 despite the fact Ž
that they are not true back-crosses the F female is not back-crossed with its male
1
. parent .
Ž .
- Three intra-strain crosses hatchery female = hatchery male : Control 1, Control 2 Ž
. and Control 3 domestic males 1 and 2 are the same as for BC1 and BC2 . These
families were used as controls to interpret the survival rates in back-crosses. Hatching took place from 29 to 30 January 1994. Approximately 300 eyed eggs were
Ž .
analysed by Guyomard 1997 in another experiment and 300 eyed eggs were sent at Ž
. 3308C = days eyed stage in Table 1 to the Mas de Merou fish farm. These 300 fry per
´
family were maintained in separate incubation tanks. The fry mortality was recorded
Table 1 Ž
. Survival rate Ts of backcrosses and control intra-strain families
Family name Breeding scheme T at 13 days T at eyed stage T at 30 days T at 2 months T at 6 months
s s
s s
s a
b
Ž .
Ž .
Ž .
Ž .
BC1 fH1=mD1
– 95
300 82.3 247
82.2 203 75.4 153
b
Ž .
Ž .
Ž .
Ž .
BC2 fH2=mD2
– 92.9
300 88 264
84.5 223 69 154
b b
Ž .
Ž .
Ž .
Ž .
Control 1 fD1=mD1
96 84.3
300 79.7 239
78.25 187 41.2 77
b b
Ž .
Ž .
Ž .
Ž .
Control 2 fD2=mD2
92 90.8
300 89.3 268
85.5 229 47.2 108
b b
Ž .
Ž .
Ž .
Ž .
Control 3 fD3=mD3
95 93
300 89.7 269
88.2 237 80.2 190
a
BC indicates the set of backcrosses analysed. F1 hybrid female are identified as fH; domesticated females Ž .
Ž and males as fD and mD respectively. Survival rates T
for progeny are indicated in percentage the raw data
s
. indicated in brackets , at different stages.
b
Ž .
From Guyomard 1997 .
from hatching until 6 months after hatching. Dead fish were counted daily and Ž
differences in survival rate between controls and back-cross were tested by ANOVA 1 .
factor, STATGRAPHICSq for Windows software . 2.2. Methods to describe protein and microsatellite Õariation
Allozymes: individuals were genotyped for the following allozymic loci: AAT-4, LDH-5, FBP-1, IDH-3 and MPI. Electrophoresis was performed on horizontal 12
Ž .
starch gel; staining buffers were similar to Beaudou 1993 . Microsatellite loci: we analysed four microsatellite loci: Strutta-12, Strutta-58 and
Ž .
Strutta-24 were obtained from a genomic library at our laboratory Poteaux, 1995 . The sequences of the primers used are:
for Strutta-24 s R: GACAGGGTCATTGATGTCATC and F: CACGGGAATACA- CACACGTG;
for Strutta-12 s R: AATCTCAAATCGATCAGAAG and F: AGCTATTTCAGA- CATCACC;
and for Strutta-58 s R: AACAATGACTTTCTCTGAC and F: AAGGACTTGAAG- GACGAC.
We also used MST-15 from the INRA laboratory: R s TGCAGGCAGACGGATC- Ž
. AGGC and F s AATCCTCTACGTAAGGGATTTGC Estoup et al., 1993 .
Total genomic DNA was isolated from a piece of muscle using the standard Ž
. phenol-chloroform methodology
Sambrook et al., 1989 . DNA from parents and back-cross individuals was amplified using the g-
33
P radioactive PCR reaction accord- Ž
. ing to the protocol in Garcia de Leon et al. 1995 . PCR products were electrophoresed
through a 6 denaturing polyacrylamide sequencing gel. Only nine of the 11 wild parents used to produce hybrids were genotyped in this
study. We assumed that the alleles observed both in the wild individuals and the hybrids are the same and originate from the wild individuals.
2.3. Statistical analysis of segregation data Single-locus segregation and joint segregation between all loci were tested with
chi-square tests for heterogeneity between loci or individuals using the general proce- Ž
. dure described by Mather Mather, 1951; May et al., 1979 . The alleles of the two loci
Ž
X X
. involved in a comparison of segregation are referred to by letters A, A , B and B . The
symbols and statistics used are detailed below. For the mating AABB = AA
X
BB
X
: a1 s observed AABB progeny;
a2 s observed AABB
X
progeny; a3 s observed AA
X
BB progeny; a4 s observed AA
X
BB
X
progeny Ž
. N s total progeny s a1 q a2 q a3 q a4 .
The chi-square tests for departure from 1:1 segregation at both loci were tested as:
2
Ž .
2 2
Ž .
2
Ž .
x s a1 q a2 y a3 y a4 rN and x s a1 y a2 q a3 y a4 rN df s 1 .
A B
To test the departure from independent segregation of the two loci involved, we used:
2
Ž .
2
Ž .
x s a1 y a2 y a3 q a4 rN df s 1 . Here, r is the fraction of non-parental geno-
AB
w Ž
. Ž
. types assuming the largest class, either a1 q a4 or a2 q a3 represents the parental
. Ž
. Ž
. x
genotypes , and r s a1 q a4 rN or a2 q a3 rN . Goodness-of-fit x
2
also was employed to test the transmission of wild versus domesticated alleles by each hybrid female.
3. Results and discussion
