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
3.1. SurÕiÕal rates Ž .
The survival rates T of progeny at different stages are reported in Table 1. There is
s
no statistical difference of survival rates between back-cross progenies, between control Ž
. intra-strain family and between back-cross and controls by ANOVA at the four early
development stages. For the survival rate at 6 months, the control intra-strain family 3 exhibited a significantly higher survival rate than the control 1 and 2. If we exclude the
results of control intra-strain family 3, survival rates between back-cross progenies and controls became significant.
The non-significant differences of survival rates at key stages led us to expect that the presumed incompatibility acting on the recombination of the genomes was weak, as
Ž .
proposed by Guyomard 1997 . This author reported that fertility appeared to be normal for both male and female F hybrids. Back-crosses are as viable as control individuals in
1
Ž .
hatchery conditions Guyomard, 1997; our data and progeny were sacrificed to 6 Ž
. months of age when the survival rates were still high Table 1 .
3.2. Single locus segregation We analysed 174 fry in BC1 and 160 in BC2. All microsatellite loci displayed
Mendelian segregation in conformity with the expected proportions of 1:1 or 1:2:1 Ž
. Table 2: 12 and 10 tests in BC1 and BC2, respectively . Although the test of all the
Ž .
data was not significant Bonferroni test, Rice, 1989 , significant non-Mendelian segre-
Table 2 Genotypes of genitors for allozyme and microsatellite loci
Mendelian segregation was tested in conformity with the expected proportions of 1:1 for underlined loci and expected proportions of 1:2:1 for double underlined loci.
Allozyme loci Microsatellite loci
AAT-4 FBP-1
IDH-3 LDH-5
MPI Strutta-12
Strutta-58 Strutta-24
MST-15 fH1
100100 100150
100100 100105
100105 1–2
1–3 1–4
1–2
a
mD1 65100
100100 100100
100105 100105
3–5 4–6
5–n 2–4
fH2 100100
100150 100130
100105 100100
1–2 1–5
3–n 2–4
mD2 100100
100150 100100
100100 100100
4–6 1–2
2–n 3–4
a
Null allele.
Ž .
gation was observed at LDH-5 for the BC2 hybrid female Table 3 . This was due to an Ž
. excess of LDH-5 90 domestic allele over LDH-5 100 ‘‘wild’’ allele. Taggart and
Ž .
Ferguson 1984 found only Mendelian segregation at this locus. Their results are not incompatible with ours, since they analysed crosses between more closely related brown
trout forms, namely ancestral and modern Atlantic populations. Their allele 100 at the LDH-5
locus originated from a wild landlocked ancestral population in the Atlantic Ž
basin and not from the Mediterranean basin as in our crosses D s 0.1 between Atlantic .
and Mediterranean forms . The second family BC2 did not show any similar deviation at this locus. However, the number of informative individuals in BC1 is much lower since
both parents are heterozygous for the same alleles at LDH-5. Hence, the aberrance of segregation at this locus should be confirmed in other hybrid parents.
LDH-5 is a diagnostic locus between Mediterranean trout and domesticated strains. It is not impossible that the allele of domesticated origin or the chromosomal region
surrounding this allele conferred an advantage in the hatchery conditions of the Ž
experiment, whereas it may have conferred a disadvantage in natural conditions Poteaux .
et al., 1998 . Indeed, selective influences at protein-coding loci have been suggested to Ž
explain the spatial pattern of gene frequencies of several species of fish Verspoor and .
Jordan, 1989; Powers et al., 1991 . Experimentally, it has been shown with poıkilo-
¨
therms that the functional differences among allozymes of numerous loci could result in Ž
different selective values Philipp et al., 1985; Powers et al., 1991; Kirpichnikov, 1992; .
Jordan et al., 1997 . In the case of brown trout, differences in enzyme affinity for the substrate were found only between the alleles of the locus LDH-5 by Henry and
Ž .
Ferguson 1985 . It has been suggested that these differences may explain the almost total colonisation of the open basins of the North Atlantic watershed by the modern
Ž .
Atlantic form Hamilton et al., 1989 . Analysis of Strutta-24 allele segregation in offspring led to the inference that the two
Ž .
domesticated males mD1 and mD2 in BC1 and BC2, respectively; Table 2 and the hybrid female fH2 in BC2 were all heterozygous for a null allele. We also inferred that
the null allele in the hybrid female fH2 came from her domesticated parent, since two of three parents from the fish farm possessed a null allele at this locus. In BC2, each
individual bred for the back-cross possessed a null allele and the allelic segregation resulting from this mating was tested. Segregation was in conformity with Mendelian
proportions and the overall test of heterogeneity on the entire set of data is not significant. The latter test showed that the null allele is transmitted as well as the other
allele.
Goodness-of-fit tests were performed for each female to compare alleles of wild and domesticated origin for allozymes, for microsatellite loci, and for both together. None of
the 13 tests was significant, and we did not observe any tendency of the hybrid female to transmit one allele rather than another.
Finally, no new mutation was detected among the meioses analysed, that is to say, 1360 constitutions of monolocus genotype analysed for BC1 and 1174 for BC2.
3.3. Joint segregation Ž .
In our experiment, joint segregation was investigated at two levels: i Segregation is assumed to be ‘‘normal’’ for the domesticated male, which is assumed to transmit each
C. Poteaux
et al.
r Aquaculture
186 2000
1 –
12
7 Table 3
Joint segregation data of loci combinations found to exhibit statistically significant linkage A, A
X
, B and B
X
represented possible gametes for the informative parents. AB, AB
X
, A
X
B, and A
X
B
X
represented each type of gametic associations for the parent
X X
Ž .
analysed. AB and A B represented parental gametic associations in each case hybrid female and domestic male . In hybrid females, alleles of Mediterranean origin were underlined; all the other alleles are of domestic origin.
X X
X X
2a 2b
2c d
e
Informative Female locus A
Female locus B Male locus A
Male locus B AABB
AA BB AABB
AA BB x
x x
r N
A B
AB
parent
U
fH1 MPI
MST-15 MPI
MST-15 13
24 21
15 0.01
0.07 3.96
0.61 73
X X
AA s100r105 BB s1r2
AA s100r105 BBs 2r4
U
fH1 St24
St12 St24
St12 38
47 47
29 0.5
0.5 4.52
0.52 161
X X
AA s1r4 BB s1r2
AA s 5r n BBs 3r5
U
fH1 St24
FBP St24
FBP 27
41 44
33 0.56
0.06 4.31
0.59 145
X X
X
AA s1r4 BB s100r150
AA s 5r n BB s100r150
UU
fH2 IDH
St24 IDH
St24 48
21 21
28 3.39
3.39 9.79
0.36 118
X X
AA s130r100 BB s n r3
AA s100r100 BBs 2rn
U
fH2 FBP
LDH5 FBP
LDH5 18
14 29
12 1.11
6.04 2.31
0.59 73
X X
AA s100r150 BB s90r100
AA s100r150 BBs90r90
U
mD1 St24
MST-15 St24
MST-15 47
28 38
47 0.62
0.62 4.9
0.42 160
X X
AA s1r4 BBs1r2
AA s 5rn BB s 2r4
U
mD2 FBP
St24 FBP
St24 21
8 15
21 0.75
0.75 5.55
0.36 65
X X
AA s100r150 BBs n r3
AA s100r150 BB s 2r n
a
Test of random segregation at locus A.
b
Test of random segregation at locus B.
c
Ž .
Test of joint segregation at locus A and B df s1 .
d
Ž .
Fraction of non parental genotypes assuming for males that the largest class represents parental genotypes .
e
All the progeny.
U
P - 0.05.
UU
P - 0.01.
allele at each locus without any selective genetic constraints. We could thus detect Ž .
linkage for the loci analysed in its offspring. ii The female parent is an F hybrid made
1
up half of genes of domesticated origin and half of genes of wild origin. Segregation was tested to verify whether there is normal recombination during meiosis between the
genomes of different origins; or alternatively, whether the females pass on associations Ž
. of alleles parental or recombined gametes advantaged rather than random combination
for these two loci. As recombination rates are much higher in females than in males in Ž
. salmonids Johnson et al., 1987 , distortions of joint segregation in females alone would
suggest a selective phenomenon when both loci are not linked in males. A total of 70 joint segregation tests were performed. In some cases, analysis was
restricted to a single family. Significant joint-segregation tests are presented in Table 3 and the entire set of tests carried out in Table 4. Non-random segregation was detected
for six locus combinations — four in the hybrid females, and two in the hatchery males Ž
. Tables 3 and 4 . According to our results, four of the six tests performed showed that
these microsatellite loci were neither linked, nor linked to the allozymic loci AAT-4 and LDH-5.
For the significant tests MST-15rMPI in fH1 and Strutta-24rIDH-3 in fH2, we were unable to conclude that these loci are independent: the tests were performed on
only one set of progeny and only one of the two tests carried out was significant. Some cases of non-random associations detected in this study may result from chance alone.
Similarly, the test result between Strutta-24 and MST-15 was significant for the BC1 domestic male, but not for the three other parents. Because the allelic segregation was
normal for this male at each locus, we can infer that the significance resulted from chance.
Significant values for Strutta-24rFBP-1 linkage were obtained in two out of three cases, for fH1 and mD2. The non-significant value for fH2 may be reflected by a higher
recombination rate in female than in male: the lower frequency of male recombination
Table 4 Summary of all pairwise examinations for joint segregation of allozymic and microsatellite loci from BC1 and
BC2 Ž
Numbers in brackets indicate the number of tests realised for each pair of loci male below and female above .
the diagonal . Statistically significant assortments are indicated by bold character: 1 for BC1 and 2 for BC2. Female
12 58
24 15
LDH-5 FBP
AAT MPI
IDH
U
Ž . Ž .
Ž . Ž .
Ž . Ž .
Ž . Male
12 1
2 1 2
2 2
– 1
1 Ž .
Ž . Ž .
Ž . Ž .
Ž . 58
2 1
1 1
1 –
1 –
U UU
Ž . Ž .
Ž . Ž .
Ž . Ž .
Ž . 24
2 2
2 2
2 1 –
1 1 2
U U
Ž . Ž .
Ž . Ž .
Ž . Ž .
Ž . 15
2 2
2 1 2
2 –
1 1 1
Ž . Ž .
Ž . Ž .
Ž . Ž .
LDH-5 1
1 1
1 2
1 –
–
U
Ž . Ž .
Ž . Ž .
Ž . Ž .
Ž . FBP
1 1
1 2 1
2 –
1 1
Ž . Ž .
Ž . Ž .
Ž . Ž .
AAT 1
1 1
1 1
– 1
– Ž .
Ž . Ž .
Ž . Ž .
Ž . MPI
1 1
1 1
– 1
1 –
Ž . IDH
– –
– –
– 1
– –
U
P - 0.05.
UU
P - 0.01.
than female recombination is in agreement with the general observation that when there is a difference in recombination rates between the sexes, the heterogametic sex displays
Ž .
lower frequencies Swanson et al., 1981 . This significant test may indicate a weak linkage between both loci, which is not detected in every case.
The significant test for Strutta-12 and Strutta-24 in BC1 may not be due to a physical linkage between these loci, since they were statistically independent for the
other individuals. One significant result in four tests may have resulted from chance since the loci are not linked, or from selection because the hybrid female transmitted
preferentially one type of her gametes, the recombined type in this case. The joint segregation at these loci has to be confirmed with the analysis of other sets of
back-crosses.
In general, if there is no difference in joint segregation between the two types of Ž
. cross back-cross and control cross in this type of experiment, then we conclude there
are no selective forces acting particularly on the hybrid individuals. In contrast, significantly higher or lower levels of linkage disequilibrium may occur in the hybrids,
which may have different causes such as lower recombination rates andror selection pressures acting against genotypes. First, when the recombination rates are significantly
higher for the hybrid individuals of a given sex, differential selection according to genotype is likely. Here, three out five tests are significant except for the female
individuals, especially the Strutta-12rStrutta-24 pair for fH1.
Secondly, if the recombination rates are significantly lower for the hybrid individuals, two hypotheses can be put forward: reduced recombination at the meiosis stage in the
hybrids or differential selection acting on genotypes. This may be the case for the most highly significant test result obtained in this study, between IDH and Strutta-24 for the
Ž .
female fH2 r s 0.36, Table 3 . Considering the origin of the alleles for these loci, we observe that the parental forms of gametes were transmitted more than the recombined
type, which would correspond to a particular selection in hatchery-reared conditions. However, other crosses with heterozygous females for these loci should be analysed to
test the hypothesis of a strong linkage between these two loci. In addition, differential post-zygotic selection acting on recombined genotypes would result in lower survival
rates in hybrids than in controls. Such a tendency is not seen here, but our data set is rather limited and variations in survival rates cannot easily be interpreted. A better
approach would be to compare these lower recombination rates with those observed in
Ž .
the same family at an earlier stage i.e., the eyed stage when the survival rate is still close to 100 and the selection hypothesis can be discarded.
Intensive restocking with hatchery-reared trout are common practices everywhere now and breeding with native stocks could lead to the loss of the genetic integrity of
aboriginal populations, by introgression and breaking up coadapted gene complexes Ž
. several articles in STOCS, 1981 . This question is of great interest for fisheries
managers. In our experiment, considering the data of survival rate and hybrids fertility, outbreeding depression did not occur. However, this experiment of crossing differenti-
ated trout genome represent a small test to study a possible breakdown of adaptation to a particular environment, here the hatchery life conditions. The reciprocal test for studying
breakdown of fitness in the wild was not tested here but it will be very interesting to analyse it in parts of half-artificial streams.
4. Conclusion
