Ž .
ences in the spermatocrit Mair, 1993 , could generate progeny with paternal genetic Ž
. Ž
material ‘false’ gynogens . This may lead to misinterpretation of the results Carter et .
al., 1991 . Furthermore, one should verify the homozygous nature of endomitotic Ž
. gynogens Bongers, 1997 and determine the degree of residual heterozygosity, through
recombination, in meiogynogenic progeny. It is possible to assess the level of recom- Ž
bination and hence the inbreeding rate by using heterozygous females Chourrout, .
1988 . Genetic markers are required to detect paternal contamination and to determine the
degree of heterozygosity in gynogenic offspring. Morphological markers can be used if Ž
. the trait is based on a recessive allele; such phenotypic markers e.g., albinism were not
available in our broodstock. Multilocus DNA fingerprinting has been used for detecting Ž
paternal contamination but it is not efficient to verify heterozygosity levels Carter et al., .
Ž 1991; Volckaert et al., 1994; Bongers, 1997 . Allozyme markers are useful Linhart et
. al., 1987 but they cannot compete with the level of polymorphism and the information
Ž .
content of single locus microsatellite markers Wright, 1992 . Furthermore, these latter markers allow biopsies.
We show the low feasibility of applying heat shocks to induce endomitosis and the presence of a few ‘false’ and relatively many spontaneous gynogens. We provide
alternative hypotheses for the high percentage of residual heterozygosity at one of the DNA markers and suggest an alternative estimation of the recombination rate. Finally,
we show evidence for male heterogametic sex chromosomes in C. gariepinus.
2. Materials and methods
2.1. Samples The catfish used in this study originate from a stock held at the Laboratory of Aquatic
Ecology, Katholieke Universiteit Leuven, Leuven, Belgium, which includes a line Ž
. Ž
originating from the Hulah Swamps Israel and a line from the Ubangui River Central .
African Republic . Fish were kept under standard conditions as detailed in Huisman and Ž
. Richter 1987 . Ovulation, egg collection, artificial fertilisation and larviculture have
Ž .
been described previously Volckaert et al., 1994 . We used in each experiment
genetically screened and electronically tagged parents belonging to two different lines. Ž
. Ž
Adults were sexed externally genital papilla and internally presence of ovaries or .
testes . 2.2. Shock experiments
The methods for UV-irradiation and heat shocking have been described in Volckaert Ž
. Ž
. et al. 1994 . We varied the shock-temperature 398C, 408C, 418C and 428C , the
Ž .
duration of the shock 1, 1.5, 2 and 3 min and especially the time of application after Ž
. activation of the egg 1–40 min; at 1 min intervals to determine the optimal shock
Ž
2
. Fig. 2. Optimal UV-irradiation dosage
Jrcm to inactivate the sperm genetically in function of the
Ž
9
. Ž .
spermatocrit value number of sperm cells per ml=10 y s 0.367q0.493log
x .
10
conditions. The first mitotic division occurred at about 35 m.a.a. at an incubation Ž
. temperature of 288C Bongers et al., 1995 . Nevertheless, we applied the shock 1 to 40
m.a.a. to compare yields in survival and heterozygosity in meio- and mitogynogens. For each experimental condition, we obtained the percentage of surviving embryos 24 and
Ž 72 h after activation. To allow for comparison between experiments using different
. females , standardised survival rates were calculated relative to the positive control
Ž . Ž
. Ž
which is a normal fertilisation Volckaert et al., 1994 . A negative control eggs
. activated with irradiated sperm but no shock applied
was included to assess the effectiveness of the irradiation. We determined the spermatocrit value of each sperm
Ž sample in order to adjust the irradiation time accordingly extended irradiation for
. samples with a high spermatocrit value; see Fig. 2 . Not all combinations of shock
Ž .
temperature and duration were analysed see Results but each experiment was carried Ž
. out in duplicate except 398C at 1 min . In total, 17 successful experiments were
performed. Embryos were kept at 288C before and after shocking. Putative endomitotic fish were reared to maturity in order to determine their sex. Survival rates were
arcsin-transformed and tested for their significance using a one-way ANOVA and
Ž .
Duncan’s multiple range test Sokal and Rohlf, 1995 . 2.3. Genetic analysis using microsatellite DNA markers
Ž .
We used five Cga01, Cga02, Cga05, Cga09 and Cga10 out of seven microsatellite Ž
. primer sets which have been developed previously Galbusera et al., 1996 and one
additional marker, Cga08, similarly developed through the production and screening of a Ž
library of short DNA fragments of C. gariepinus. Cga08 GenBank accession number .
U30869 has the primer sequences F-CATGAGCCAGACACCATTCCC and R-TTTC- Ž
. CATGTGTTGGTCCTTGC, with a GT
repeat sequence; PCR products have a size
11
Table 1 Ž
. Survival rate percentage relative to the positive control, after 24 and 72 h, rp24 and rp72, respectively of
Ž .
Ž .
positive pos and negative neg control, meiogynogenic and mitogynogenic progeny in each experiment Ž
. m.a.a.s minutes after activation — other shock conditions are: temperature in 8C_duration in minutes
m.a.a. rp24
rp72 rp24
rp72 rp24
rp72 rp24
rp72 rp24
rp72 39_1
exp1 39_1,5
exp2 39_1,5
exp3 39_2
exp4 39_2
exp5 1
99 2
14 16
2 84
1 14
6 4
5 7
6 3
90 33
7 19
38 25
5 4
83 66
18 28
7 43
7 5
86 73
21 38
2 56
37 5
6 78
1 57
11 37
5 74
27 7
80 51
2 37
71 3
8 87
57 5
38 71
14 9
94 64
3 31
3 54
21 10
91 47
8 46
60 6
63 11
90 63
3 54
59 9
3 12
96 93
13 50
3 51
5 18
13 94
99 8
47 73
58 14
84 81
6 42
7 76
77 15
93 70
5 52
3 59
9 43
16 84
72 40
83 6
39 3
17 90
56 3
61 2
42 44
18 84
87 2
73 100
69 19
88 60
2 68
48 49
5 20
85 1
66 38
41 3
21 86
67 2
30 4
63 65
22 88
47 3
37 36
7 28
5 23
82 63
23 2
10 7
24 76
60 10
8 6
25 52
60 3
26 74
35 2
27 86
6 28
90 37
2 11
15 17
29 88
27 30
87 15
66 3
26 58
31 92
42 32
89 5
26 16
20 33
86 50
4 34
83 20
36 40
44 3
35 88
50 15
5 54
36 71
19 35
3 19
38 37
11 12
20 22
38 23
25 4
24 3
18 5
39 72
9 2
40 73
40 13
5 3
41 64
43 pos
97 97
50 27
92 92
80 67
86 100
pos 103
99 101
98 106
108 123
132 118
98 neg
98 1
60 2
68 4
60 32
neg 93
68 52
4 58
117
Ž .
Table 1 continued m.a.a.
rp24 rp72
rp24 rp72
rp24 rp72
rp24 rp72
rp24 rp72
40_1 exp6
40_1 exp7
40_1,5 exp8
40_1,5 exp9
41_1 exp10
1 44
7 10
8 5
1 10
9 2
71 13
27 2
5 3
19 4
21 23
3 92
36 56
7 52
5 25
7 35
3 4
101 31
51 6
52 4
36 6
38 2
5 85
41 50
4 69
5 39
4 36
6 93
15 57
4 59
5 30
3 31
7 72
9 38
1 50
3 15
31 8
103 17
47 64
6 12
2 27
9 93
24 26
5 66
3 31
10 89
2 46
3 62
7 1
31 11
86 12
37 9
51 1
7 33
2 12
92 20
55 29
14 2
29 4
13 87
8 36
13 1
7 44
2 14
86 17
38 2
10 9
38 2
15 86
2 3
7 58
16 94
21 12
3 8
1 27
17 64
2 12
7 38
18 87
19 23
1 46
1 6
36 19
85 10
32 2
45 6
10 29
2 20
74 13
24 6
24 4
6 2
31 21
93 20
43 5
6 4
24 22
86 18
34 2
3 8
16 23
81 4
37 8
2 24
91 13
34 5
2 12
1 25
84 11
22 12
6 1
26 71
14 11
33 3
27 44
1 36
3 28
42 11
5 16
1 1
29 38
2 27
1 4
30 65
6 13
3 31
1 7
31 71
3 43
3 11
1 32
95 21
16 33
1 11
1 10
33 81
8 16
12 12
34 109
23 32
2 12
7 1
13 35
96 24
4 21
2 23
1 36
97 21
19 21
2 13
3 37
77 16
16 10
12 17
38 62
16 25
6 27
9 9
39 35
8 19
3 19
12 7
40 26
16 5
15 7
41 22
4 15
42 4
3 pos
104 108
134 145
99 102
104 101
95 85
pos 94
94 64
54 101
97 97
99 107
115 neg
113 31
46 75
2 76
2 42
1 neg
109 30
73 5
89 1
72 15
37 ?
? continued on next page
Ž .
Table 1 continued m.a.a.
rp24 rp72
m.a.a. rp24
rp72 m.a.a.
rp24 rp72
m.a.a. rp24
rp72 41_1
exp11 40_1–2
exp12 40_1–2
exp13 41_2
exp14 1
17 2
2 20
28 10
1 51
50 2
42 20
37 2
21 16
5 2
60 26
3 53
3 25
24 22
24 3
50 29
4 68
30 19
23 6
4 37
7 5
83 2
32 34
24 5
13 6
63 34
28 26
10 6
7 73
36 61
27 9
8 3
8 79
7 38
57 1
28 3
10 2
9 60
2 40
42 29
3 4
15 3
10 71
1 42
5 30
17 5
20 14
3 11
64 1
44 4
31 16
24 12
71 2
50 17
32 19
28 13
74 3
55 45
34 57
9 30
1 14
79 60
35 44
32 2
15 61
1 2
39 25
36 24
34 18
16 73
20 12
37 5
7 36
17 71
25 1
38 9
38 18
67 1
30 2
39 40
19 65
1 32
16 40
pos 100
100 20
66 34
13 20
8 neg
80 21
69 36
32 21
11 neg
81 22
66 38
43 22
23 56
40 8
23 24
26 42
1 24
25 1
44 26
26 1
50 6
27 27
2 55
26 28
3 28
7 1
60 29
29 23
pos 122
124 30
30 36
pos 79
75 31
4 5
31 55
neg 77
1 32
32 80
neg 65
34 15
33 78
1 35
7 34
82 1
36 3
35 61
37 36
73 2
38 37
52 2
39 38
25 40
39 36
1 pos
91 78
40 33
pos 105
122 pos
97 93
neg 65
11 pos
103 107
neg 41
4 neg
88 1
neg 42
Ž .
range of 158–180 bp and optimal PCR conditions are 628C annealing temperature and 1.5 mM MgCl . Each primer set amplifies a specific microsatellite locus in a PCR. The
2
Ž .
Table 1 continued m.a.a.
rp24 rp72
m.a.a. rp24
rp72 m.a.a.
rp24 rp72
40,5_2 exp15
40_1 exp16
39_1,5 exp17
1 113
98 20
34 27
21 11
2 2
110 82
21 59
39 22
10 5
3 87
1 22
26 15
23 8
2 4
84 23
16 11
24 4
5 114
24 46
30 25
8 6
164 26
23 11
26 2
3 8
168 27
21 8
27 6
2 10
164 28
40 19
28 2
15 40
29 25
5 29
9 20
57 1
30 12
30 13
7 24
1 31
20 15
31 19
2 26
32 15
6 32
13 3
28 3
34 31
26 33
35 30
25 35
25 19
34 39
32 9
36 12
4 35
15 3
34 20
37 39
27 36
16 36
40 38
41 18
37 11
38 14
39 41
25 38
7 2
40 40
17 18
39 10
3 24
pos 100
100 40
2 3
26 neg
104 62
pos 102
121 28
pos 98
75 30
1 neg
46 2
32 neg
31 2
34 36
pos 100
100 neg
169
genomic DNA of fin-clips or whole larvae of about 1 week old was prepared using Ž
phenol–chloroform extraction or by boiling for 2 h in a 10 Chelex solution Resin .
100, Bio-Rad Laboratories, Hercules, CA . The DNA was amplified according to Ž
. Galbusera et al. 1996 .
Three methods are used to visualise these PCR products after electrophoresis depending on the resolution needed to distinguish between alleles. Random labelling
35
Ž with
S or end labelling with a fluorescent molecule Fluos-Phosphororamidite from .
Eurogentec, Seraing, Belgium is used to visualise the PCR-products which are sepa- rated on a 6 PolyAcrylamide-gel. EtBr is applied after separation of the products on a
4 NuSieve GTG agarose-gel. In order to check whether there is any paternal contamination in the gynogenic
progeny caused by incomplete UV-irradiation, broodstock fish were selected with different alleles for at least one microsatellite locus. Heterozygous females were chosen
in order to measure recombination rates. Because the strains used have been domesti- Ž
. cated for many years about 10 to 20 generations , few females were heterozygous at
Ž .
several loci Volckaert and Hellemans, 1998 .
3. Results
