Ž .
Population genetic models have been developed review in David, 1998 explaining Ž .
Ž i the gene heterozygosity–fitness correlation Turelli and Ginzburg, 1983; Smouse,
. Ž .
1986; Mitton, 1993; Deng and Fu, 1998 , and ii neutral marker heterozygosity–fitness Ž
. correlation due to association with selected genes deleterious or overdominant in small
Ž .
populations Ohta, 1971, 1973; Ohta and Kimura, 1970, 1971; Zouros, 1993 or in Ž
populations with partial inbreeding Ohta and Cockerham, 1974; Strobeck, 1979;
. Charlesworth, 1991; Zouros, 1993 . Nevertheless, as the neutral status of allozymes is
questionable, the debate was at first concentrated on the comparison of two hypotheses: the ‘‘direct overdominance’’ hypothesis that treats allozymes as the causative agent of
Ž the correlation, versus the ‘‘associative overdominance’’ hypothesis Ohta, 1971; Zouros
. et al., 1980 where neutral markers are indicators of genetic conditions responsible for
Ž .
the correlation. More recently, David et al. 1995 proposed a further partition of the ‘‘associative overdominance’’ hypothesis into the ‘‘local effect’’ hypothesis where
markers reflect variation at fitness loci localized in their chromosomal vicinity, and the ‘‘general effect’’ hypothesis where homozygosity at marker loci correlates with overall
Ž .
genomic homozygosity in partly inbred populations also see David, 1998 . However, bottlenecks enhance linkage disequilibrium as well as inbreeding, and the partition of
associative overdominance into local and general effects is presently not well understood Ž
. Zouros, 1993 .
The development of a new class of genetic marker loci exhibiting variation in non-coding DNA sequences, and which are most unlikely to be anything other than
selectively neutral, allows a new approach to this problem, as only ‘‘associative Ž
overdominance’’ can apply in this case Zouros and Pogson, 1994; Bierne et al., 1998; .
Ž .
Pogson and Fevolden, 1998 . Pogson and Zouros 1994 found in the scallop Pla- copecten magellanicus, a MLH-growth correlation with seven allozyme loci, while a set
Ž .
of eight nuclear DNA loci restriction fragment-length polymorphisms, RFLPs failed to produce a significant correlation. A second test of the associative overdominance
Ž .
hypothesis using DNA markers was published by Pogson and Fevolden 1998 , showing a positive correlation between growth rate and RFLP marker heterozygosity at 10 loci
within a single population of Atlantic cod, Gadus morhua. Use of the three microsatellite loci recently developed in the shrimp P. stylirostris
Ž .
Ž .
Vonau et al., 1999 , and a heterologous locus from P. Õannamei Garcia et al., 1996 , allowed us to study the MLHrgrowth correlation at presumably neutral genetic markers,
within closed populations that had been maintained at small effective sizes for 22 or 24 generations. We explore the possibility of using marker-associated heterosis to evaluate,
Ž .
with Ohta’s 1971, 1973 formula, the residual inbreeding load in such inbred popula- tions with unknown pedigrees.
2. Materials and methods
2.1. History of the populations studied The last import of P. stylirostris wild founders from Mexico to two IFREMER
Ž .
Ž .
experimental hatcheries, located in Tahiti COP and in New Caledonia SASV , took place in 1980. The exact number of founders is, unfortunately, unknown. As the shrimp
Ž .
industry developed more quickly in New Caledonia, SASV used more breeders N f 100 Ž
. at each reproduction than the COP experimental hatchery N f 20 . However, it cannot
be ruled out that these populations went through stronger genetic bottlenecks than these optimistic values might suggest. In 1988, the COP stock became extinct, and was
reinitiated following the import of a part of the SASV production at the post-larvae stage. Since 1989, no transfer of breeders has been made between the two hatcheries. A
genetic improvement program began at the COP facilities in 1992. We used one stock
Ž from this program, where the number of breeders selected was 24 16 males and eight
. females
at each generation. This led to an effective population number N s
e
Ž . Ž
. 4 N N r N q N s 21.33, where N
is the number of males and N the number of
m f
m f
m f
Ž .
females. In Tahiti, a second hatchery Ecloserie Polyvalente Territoriale, EPT was constructed in the late 1980s. This hatchery depended on the COP breeders until 1993,
when it started to use its own breeders only. The presumed demographic histories of the COP and EPT populations are summarized in Fig. 1.
2.2. Sampling A first sampling of 30 individuals from the EPT population was made without
Ž .
Ž .
phenotypic characterization EPT-g in late 1996 Vonau et al., 1999 . A second
sampling, including sex determination and weighing of 48 individuals, was done two Ž
. generations later, in late 1997 EPT-g . Two further samplings, each of 30 individuals,
2
Ž were made at two different times in early 1997 in the same COP population t : mean
1
. weight s 26.8 g, t : mean weight s 43.1 g . Each individual was sexed, weighed, and
2
sampled for hemolymph. The hemolymph was diluted in an anticoagulant, Alsever Ž
solution pH 7 30 mM trisodium citrate, 115 mM glucose, 338 mM NaCl, 10 mM .
EDTA for DNA extraction. All animals sampled at the same time belonged to the same cohort, and thus had the same age.
2.3. DNA extraction, polymerase chain reaction PCR procedures and electrophoresis Ž
. The fast Chelex extraction method modified from Singer-Sam et al., 1989 was used.
Ž .
Hemolymph samples corresponding to one million cells were centrifuged at 6000 = g
Fig. 1. Presumed demographic and inbreeding histories of the COP and EPT hatchery-propagated P. stylirostris stocks. D F is the expected inbreeding increment per generation, N is the population size, and f is
the expected mean inbreeding coefficient of the stock when sampled. Estimated F values between pairs of
st
populations and their significance level, are specified:
U
P - 0.05,
UU
P - 0.01,
UUU
P - 0.001.
Ž for 2 min. The supernatant was saved and 500 ml of 5 chelating resin Chelex, Biorad,
. Ž
. Richmond, VA, USA and 5 ml of proteinase K 10 mgrml were added to each sample
tube. The mixtures were shaken and heated for 4 h in an incubator at 558C, then vortexed, heated again at 1008C for 15 min, vortexed a second time and centrifuged at
10,000 = g for 2 min. The supernatant was stored at y208C until it was used as template for PCR.
Ž Three P. stylirostris microsatellite loci, namely Pstyli.05 tandem repeat: dinucleo-
. Ž
tide, GenBank accession number: AF097487 , Pstyli.19 tandem repeat: mono and
. Ž
dinucleotide, GenBank accession number: AF097488 and Pstyli.09 tandem repeat: . Ž
dinucleotide, GenBank accession number: AF097489 full details are given in Vonau et
. al., 1999 and a heterologous locus from P. Õannamei, called Vanna.02, were used.
Vanna.02 primers were designed from the published sequence of a microsatellite Ž
. Garcia et al., 1996 in two flanking open reading frames which are thought to be highly
conserved across taxa: Primer 1 s 5
X
-GGATAACACTGATTAGATATCACGG-3
X
and Primer 2 s 5
X
-TAAATGTACACGAGATGCACC-3
X
. This marker was screened only in the EPT samples.
All other heterologous microsatellite loci which were tried, from P. monodon Ž
. Ž
. Brooker et al. in press and P. japonicus Moore et al., 1999 , and for which primers
were designed a priori in non-coding sequence, failed to amplify. This is in accordance Ž
. Ž
with the results of Moore et al. 1999 , and with two mtDNA analyses Palumbi and .
Benzie, 1991; Baldwin et al., 1998 which revealed that morphological similarity in penaeids masks very large genetic differences.
A PCR mixture of 12 ml, containing 0.5 mM of each primer, 2 mM MgCl , 0.2 mM
2
Ž of each dNTP and 0.25 unit of red Goldstar DNA polymerase Eurogentec, Liege,
`
. Belgium , was added to 3 ml of DNA solution during the first 2 min–948C denaturing
Ž .
Ž stage of the PCR program Hot Start . Thirty PCR cycles 30 s at 948C, 1 min at the
. optimum hybridization temperature, 1 min at 728C were run in a PTC 100 pro-
Ž .
grammable thermal controller MJ Research, Waltham, MA, USA . PCR products were Ž
electrophoresed through 6 polyacrylamide gels acrylamide: bisacrylamide, 29:1, 7 M .
urea using 0.5 X Tris-Borate-EDTA buffer. The gels then were silver-stained according Ž
. to Bassam et al. 1991 .
2.4. Genetic diÕersity analyses Ž
. Allele frequencies, observed and unbiased expected heterozygosities Nei, 1978 , and
Ž .
Ž .
Ž Weir and Cockerham 1984 estimation of Wright’s 1951
F-statistics F , which
is
Ž .
measures within-population departures from Hardy–Weinberg equilibrium HWE , and .
F , which quantifies genetic differentiation between pairs of populations were com-
st
Ž .
puted using the G
ENETIX
3.3 software Belkhir et al., 1996–1999 . Departure of F and
is
F from zero was tested using the permutation approach provided by this software.
st
Where more than one test was performed, the probability threshold level was adjusted according to the Bonferroni procedure.
2.5. Variance of allelic frequencies Estimates of variance of allele frequencies were made according to the theory of
Ž .
selectively neutral alleles in finite populations Waples, 1989 . Estimates of temporal
Ž .
variance of allelic frequencies, F Nei and Tajima, 1981 , were computed for each pair
c
of samples and compared to expectations under the presumed demographic parameters. Ž
. When applicable the same population at two different times , per-generation effective
population sizes, N , also were estimated.
e
2.6. Linkage disequilibrium Ž
.
2
Weir’s 1979 two-locus correlation coefficient, R , was estimated using Black and Ž
. Krafsur 1986 method and its departure from zero was tested using a permutation
approach provided by the procedure LINKDIS in Genetix. The expected correlation corresponding to the effective sizes estimated for the various presumed demographic
histories and the assumption of no physical linkage between loci were calculated using
Ž .
Weir and Hill’s 1980 formula:
2 2
1 y c q c
1 1
Ž .
2
E R s
q q
1
Ž .
Ž .
2 N c 2 y c n
2 N c 2 y c
Ž .
Ž .
e e
Ž where c is the recombination rate between the two loci here, we assumed they were
. Ž
unlinked and used 0.5 , n is the sample size, and N is the effective population size we
e
have taken the harmonic mean of the presumed real population sizes over the 22 or 24 .
generations . The last term in the equation is a correction for monogamy. The haploid Ž
. chromosome number in P. stylirostris is 46 Nakaura et al., 1988 , so the probability of
a physical linkage between a pair out of three microsatellites picked up at random is small.
2.7. MLH r growth correlation Ž
Since no difference in weight was detected between males and females F s 0.22, df s 29, P s 0.64 for COP-t ; F s 2.13, df s 29, P s 0.155 for COP-t ; F s 0.17;
1 2
. df s 47; P s 0.68 for EPT-g , the data for sexes were pooled. To facilitate compar-
2
isons among samples and to obtain a normally distributed variable, individual weight was log -transformed and expressed in standard deviation units from the sample mean
e
Ž .
standardized deviates . The locus-specific effects of heterozygosity on growth were expressed as:
d s M y M
2
Ž .
SH et SHom
M and M
being the mean standardized growth in heterozygotes and homozy-
SH et SHom
Ž .
gotes, respectively Gaffney, 1990; Bierne et al., 1998 . A homozygote depression for growth was calculated using the formula:
W
Ho m
d s 1 y 3
Ž .
W
Het
where W is the mean weight for homozygotes and W
the mean weight for
Ho m Het
heterozygotes, with the aim to use homozygote growth rate depression to estimate inbreeding load. The effect of heterozygosity versus homozygosity was tested using a
simple ANOVA. For locus Vanna.02 in the EPT population, only one individual appeared heterozygous, but it was the largest of all individuals in the sample. The
standardized deviate of this individual was two-tailed tested against the normal distribu- tion. For the COP population, a test of homogeneity of variance between the two
Ž samples, t and t , was performed. As the homogeneity was rejected F s 0.39; df s 29;
1 2
. P s 0.006 , data were not pooled, but unilateral P-values for the different tests were
Ž combined sample by sample according to Fisher’s procedure Sokal and Rohlf, 1996, pp.
. 794–797 . P-values also were combined locus by locus.
Standardized deviates were plotted on MLH and the relationship between the two parameters was expressed as the product-moment correlation r. The significance of r
Ž .
was tested by a t-test Sokal and Rohlf, 1996, pp. 574–575 . To combine the two Ž
. samples in the COP population, the average r after z-transformation was computed
Ž after homogeneity among the two correlation coefficients was tested Sokal and Rohlf,
. 1996, pp. 581–582 .
3. Results
