JOURNAL OF THE Vol. 40, No.

5
WORLD AQUACULTURE SOCIETY October, 2009

Parentage Determination and Effective Population Size Estimation

in Mass Spawning Pacific Oyster, Crassostrea gigas, Based
on Microsatellite Analysis
Ronghua Li, Qi L1 , and Ruihai Yu
Fisheries College, Ocean University of China, Qingdao 266003, China

Abstract
Seven high polymorphic microsatellite loci were used to determine the pedigrees in a mass spawning
of Pacific oyster, Crassostrea gigas, and to estimate the genetic variability between broodstock and
offspring. Parental assignment was performed on a total of 155 individuals, including 141 offspring,
8 candidate mothers, and 6 candidate fathers. The assignment results of real offspring were generally
in agreement with simulation with a success rate over 99% using only six of these loci. The
allelic diversity and observed heterozygosity (Ho ) exhibited similarity between parents and offspring
populations, but the expected heterozygosity (He ) had a significant decrease in offspring. Although all
the males and females contributed to the next generation, the variances of reproductive success and
unequal sex ratio resulted in a decline in effective population size (Ne = 11.42). The inbreeding rate
of this small-scale, mass spawning population was estimated at approximately 16.5% per generation.
This gave us an insight that when designing breeding programs based on mass spawning for future
oyster cultivation generations, the higher inbreeding and lower effective population size should be
considered.

The high fecundity and survival rate of some
aquaculture organisms in a culture environment
ensure that a small number of stocks are used
for production. The restricted number of breed-
ing individuals used in aquaculture operations
can lead to random drift in gene frequencies
between generations (Hedgecock and Sly 1990)
and a loss of genetic variability in a population
(Primack 1998).
Reductions in genetic variation showed nega-
tive effect on the commercially important traits
such as growth rate and fitness in some aquatic
animals (Koehn et al. 1988; Danzmann et al.
1989). The hatchery-produced juveniles tend
to have reduced genetic variability, measured
as number of alleles and heterozygosity, when
compared with wild or broodstock populations,
because of the small effective number of par-
ents (Allendorf and Phelps 1980; Ryman and
Stahl 1980; Cross and King 1983). Increasing
the effective number of the broodstock is an
effort to prevent the reduction of genetic diver-
sity. However, the effective number of parents
was 30% less than the actual number of broods
1 Corresponding author.
© Copyright by the World Aquaculture Society 2009
in black and red sea bream (Taniguchi et al.
1983; Sugama et al. 1988), and 57% less in
Japanese flounder (Hara and Sekino 2003).
The effective number of breeders or the
effective population size (Ne ) is affected by sev-
eral factors such as a small number of breeders,
unequal sex ratios, unequal contribution of par-
ents, and family size variations (Lannan 1980;
Gaffney and Scott 1984; Gall 1987; Hedgecock
and Sly 1990; Gaffney et al. 1992; Hedrick
2000; Koljonen et al. 2002). Ne is also essential
information to evaluate the rate of inbreeding
(Gall 1987). The high inbreeding rate will pro-
duce negative effects such as the expression of
lethal or lower fitness recessive genes caused
by increased homozygosity, inbreeding depres-
sion, and reduction of genetic variance (Fal-
coner 1989). Pedigree tracing technique allows
the direct quantification of Ne in hatcheries with
random mating (Perez-Enriquez et al. 1999).
Because of their high level of polymorphism
and codominant characterization, microsatellite
markers have been acknowledged as an efficient
tool for examining pedigree structure of hatch-
ery populations (Herbinger et al. 1995; Norris
et al. 2000; Sekino et al. 2003).

667
668 LI ET AL.
The Pacific oyster, Crassostrea gigas, sup-
ports an important molluscan aquaculture indus-
try along the coastal areas of China. The pro-
duction of oysters reached 3.82 × 106 metric
tons in 2004 in China, accounting for 33.1%
of total marine molluscan yield (Department of
Fisheries 2005). The Pacific oyster accounts for
about one-third of the total oyster production.
In China, the Pacific oyster seeds are pro-
duced exclusively in hatcheries by mass spawn-
ing. Because of the high fecundity of this
species, relatively few parents are required
to generate sufficient offspring without con-
cerns that genetic variability would decrease
in farmed populations. To avoid genetic degen-
eration in hatchery-produced seed, maintaining
effective population size and avoiding inbreed-
ing are a couple of measures generally recom-
mended. Thus, the knowledge of the pedigree
structure and the size of the broodstock that is
effectively contributing to the next generation
are of particular importance.
This study was conducted to assess the abil-
ity of microsatellites DNA markers in parent-
age assignment of mass spawn-produced Pacific
oyster, and the consequence of individual repro-
duction success on effective population size
and inbreeding rate are discussed. In addition,
the difference in genetic variability between
broodstock and offspring generations was esti-
mated.
Materials and Methods
Broodstock and Offspring
The broodstock of 2-yr-old C. gigas was
collected from a local oyster farm and con-
ditioned for 2 wk before spawning at Haili
Hatchery, Shandong Province, China. In May
2006, six males and eight females were ran-
domly selected from the broodstock population
and synchronously induced to spawn through
stimulations of air exposure and warmed seawa-
ter. Twenty-four hours after fertilization, sam-
ples of D-larvae were collected and preserved
in 100% ethanol at 4 C.
DNA Extraction and Microsatellite Analysis
Genomic DNA was extracted from adduc-
tor muscle of parental oysters and whole D-
larvae. DNA was isolated from adductor mus-
cle of the individual broodstock following Li
et al. (2006). Larval DNA was prepared by the
Chelex-modification extraction method as pre-
viously described by Li et al. (2003).
Seven microsatellite markers: L10, L48
(Huvet et al. 2000), Cg49, Cg108 (Magoulas
et al. 1998), ucdCg10, ucdCg14, and cmrCg143
(McGoldrick et al. 2000) were selected for
genotyping all parental oysters and offspring
based on their level of heterozygosity and
genotyping reliability. Polymerase chain reac-
tions (PCRs) were performed using 10 μL vol-
umes of 0.25 U Taq DNA polymerase (Takara,
Japan), 1 × PCR buffer, 0.2 mM dNTP mix,
1 μM of each primer set, 1–2 mM MgCl2 , and
about 100 ng template DNA for parental oys-
ters (1 μL of total DNA extracted for larvae).
The PCR cycling conditions for all loci of
broodstock were performed as follows: 3 min
at 94 C; 35 cycles of 1 min at 94 C, optimal
annealing temperature (55–62 C) for 1 min,
72 C for 1 min per cycle; followed by a final
extension at 72 C for 5 min. In order to improve
the amplifications from larvae, 40 PCR cycles
were run for all loci: 3 min at 95 C followed
by seven cycles of 1 min at 94 C, 30 sec at the
annealing temperature, and 30 sec at 72 C, fol-
lowed by 33 cycles of 30 sec at 94 C, 30 sec
at annealing temperature, 30 sec at 72 C, with
a final extension of 5 min at 72 C. Amplifica-
tion products were resolved via 6% denaturing
polyacrylamide gel and visualized by silver-
staining. A 10-bp DNA ladder (Invitrogen, Inc.,
Carlsbad, CA, USA) was used as a reference
marker for allele size determination. To avoid
inaccuracy in scoring because of the differ-
ences in gels, two control DNA samples were
included in each set of samples for each gel.
Statistical Analysis
The simulation program of CERVUS v. 2.0
(Marshall et al. 1998) was first used to estimate
the efficiency and the number of loci needed
for unambiguous parentage assignment of the
 PARENTAGE AND EFFECTIVE POPULATION SIZE ESTIMATION IN OYSTER
oyster. Simulations were based on hypothetical
parent and offspring genotypes that were cre-
ated using allele frequency information from
the seven microsatellite loci (Li et al. 2006).
Parental genotypes were generated assuming
Hardy–Weinberg equilibrium (HWE) and no
linkage between loci. Progeny genotypes were
generated from all parental male and female
genotypes assuming no mutation or transmis-
sion error between parents and their progeny.
This program calculates the likelihood ratios
for paternity/maternity inference and defines
a statistic  as the difference in log likeli-
hood ratios (LOD score) between the two most-
likely parents. Then, when working with real
data, each descendant is assigned to the most-
likely parent (maximum LOD with significant
 value).
The polymorphic information content (PIC)
and two exclusion power for every locus were
also calculated by this program. The power of
each locus and the combined probability over
loci to exclude a false candidate parent knowing
only the genotypes of the offspring was termed
as Excl 1 probability, and that one additionally
knowing the genotype of one parent was termed
as Excl 2 probability. The combined probability
of exclusion over loci was conducted by adding
loci from the most informative to the least infor-
mative one according to their PIC values. Exact
tests for conformance to HWE and linkage
disequilibrium at each locus were performed
using the program GENEPOP v. 3.4 (Raymond
and Rousset 1995) to obtain precise theoretical
power of exclusion. The sequential Bonferroni
correction was used for multiple tests across
loci (Rice 1989). MICRO-CHECKER v. 2.2.3
(http://www.microchecker.hull.ac.uk/) assessed
the probability of null alleles at each of the
seven microsatellite loci.
Parentage analysis was conducted using the
likelihood-based approach in CERVUS v. 2.0.
The critical  score calculated by the sim-
ulation module was used here to assign the
offspring to the most-likely candidate parent.
The parameters for this simulation run were
as follows: 10,000 replication cycles, a pool
of 14 candidate parents, 100% of the candi-
date parents sampled and genotyped, a default
669
typing error rate of 1% was used, and assign-
ment to parents was performed in a two-step
analysis from a pool of candidate sire and dam
data. Because all possible parental genotypes
were known, it was implied that a genetic mis-
match between offspring and the known parent
genotype was because of either miss scoring or
mutation. Cumulative assignment success was
also evaluated based on parent and offspring
genotypes.
Because of the likelihood of genotyping
errors, we used another program PAPA v. 2.0
(Duchesne et al. 2002) that has an algorithm
that can account for genotyping errors to per-
form a parental pair allocation and compared
between the programs.
Levels of genetic variation for broodstock
and offspring populations were assessed, as the
number of alleles per locus, expected heterozy-
gosity (He ), and observed heterozygosity (Ho )
were calculated using CERVUS 2.0 (Marshall
et al. 1998). All correctly allocated offspring
were included in this and in the subsequent
analyses. Significance was tested using the
Wilcoxon matched-pairs signed-rank test (Zar
1999) implemented with SPSS v. 14.0 software
(SPSS Inc., Chicago, IL, USA).
Parental contributions to reproduction were
represented by male/female reproductive suc-
cess, which was calculated for each male/female
as the proportion of offspring from a single
spawning sired or dammed by that male/female.
Chi-square analyses implemented with SPSS v.
14.0 were used to test for the differences in
male/female reproductive success.
As an estimator of male sperm competi-
tion, the genetically effective paternity fre-
quency was calculated following
 Bekkevold
et al. (2002) as EPF = 1/ pi2 , where pi is
the proportion of offspring sired by male “i” in
a given clutch. The same formula was also used
to compute the genetically effective maternity
frequency (EMF).
The effective population size was first cal-
culated for unequal sex ratio after Crow and
Kimura (1970) as: Ne = 4Nm Nf /(Nm + Nf ),
where Nm and Nf are actual number of males
and females. Then the Ne was recalculated
based on the differences in reproductive success
670 LI ET AL.

Table 1. Numbers of alleles (k), polymorphic
information content (PIC), and probabilities of exclusion
based either on genotype of no parent known (Excl 1) or
one parent known (Excl 2) for the seven microsatellite loci
analyzed in the present work.
where ci is the fractional contribution of each
parent, cm is the average fractional contribu-
tion of males, 1/(2Cm ), and cf is the average
fractional contribution of females, 1/(2Cf ).

Locus k PIC Excl (1) Excl (2)

Results
Cg49 29 0.952 0.831 0.907
cmrCg143 17 0.888 0.656 0.792 Computer Simulations
Cg108 27 0.952 0.829 0.907
ucdCg14 38 0.950 0.826 0.904 Summary statistics for the seven markers
ucdCg10 22 0.936 0.780 0.876 used in the simulations of the hypothetical oys-
L10 46 0.971 0.894 0.944 ter breeding population are given in Table 1.
L48 32 0.951 0.828 0.906
The allele number of these loci ranged from a
Average 30.1 0.943 0.806 0.891
minimum of 17 for cmrCg143 to a maximum
of 46 for L10 with an average of 30.1 and the
mean PIC of 0.943. The large variability exhib-
of individuals, where Nem and Nef are the ited by the seven microsatellite loci analyzed
effective number of males and females, respec- leads to a high power for parentage assignment.
tively. Nem and Nef were estimated follow- Probabilities of exclusion per locus ranged from
ing Lande and Barrowclough (1987), Nem = 0.656 to 0.894 when only information of off-
(Nm k m − 1)/[k m + (σ 2km /k m ) − 1], where Nm spring was available (Excl 1) and from 0.792
is the actual number of males; k m is the average to 0.944 when one parent was known (Excl 2).
number of offspring sired by an individual male Combined assignment success of the seven
and σ 2km is the variance of km . The same for- loci in the hypothetical population with a two-
mula was used to compute the effective number step analysis was calculated by adding loci from
of females. the most informative to the least informative
The rate of inbreeding, F , was calculated one according to their PIC value (Fig. 1).
by Brown et al. (2005) after using the following Simulations showed that four loci were required
formula: to assign 90% of progeny to both parents and
1  2 1 1 five loci to assign 99%. The total assignment
F = c − (cm )2 − (cf )2 (1) success would be over 99% if the loci were up
2 parents i 4 4
to six.

1.2
0.99 1.00 1.00
Assignment success rates

1.0 0.90
0.98 1.00 1.00
0.8 0.88
0.61
0.6
0.45
0.4 simulation data
real data
0.2
0.03
0 0.07
0
L10 Cg49 Cg108 L48 Cg14 Cg10 Cg143
Loci added

Figure 1. Cumulative assignment success rates by CERVUS v. 2.0 of 10,000 simulated offspring to their 14 hypothetical
candidate parents and real genotype date sampled in a strict level of 95% confidence interval, adding loci from the most
to the least PIC one.
 PARENTAGE AND EFFECTIVE POPULATION SIZE ESTIMATION IN OYSTER 671

Table 2. Genetic variability of the broodstock and offspring of Pacific oyster, Crassostrea gigas.

Locus

Population Cg49 cmrCg143 Cg108 ucdCg14 ucdCg10 L10 L48 Mean

Broodstock
Sample size 14 14 14 14 14 14 14
Alleles range (bp) 132–182 140–162 122–174 198–232 150–234 114–214 90–172
No. of alleles 14 11 14 10 15 15 14 13.3
Expected heterozygosity 0.929 0.918 0.937 0.897 0.947 0.950 0.937 0.931
(He )
Observed heterozygosity 0.857 0.714 0.929 0.357 0.357 1.000 0.857 0.724
(Ho )
Offspring
Sample size 128 129 128 110 127 124 136
Alleles range (bp) 132–182 140–162 122–174 198–232 150–234 114–214 90–172
No. of alleles 14 11 14 10 15 15 14 13.3
Expected heterozygosity 0.901 0.857 0.868 0.882 0.915 0.903 0.915 0.892
(He )
Observed heterozygosity 0.945 0.853 0.922 0.664 0.638 0.935 0.934 0.842
(Ho )

Analyses from MICRO-CHECKER showed
probable evidence of null alleles (general excess
of homozygotes) at ucdCg10, ucdCg14, and
cmrCg143, but no evidence of “stutter peaks”
which may have resulted in scoring errors or
large allele dropout at any locus. The three
loci also evidenced departure from HWE pro-
portions before Bonferroni correction, with two
of them (ucdCg10, ucdCg14 ) significant after
Bonferroni correction. There was no significant
pair-wise linkage disequilibrium between any
loci, which was in accordance with independent
segregation required by CERVUS.
Parentage Analysis and Genetic Variability
Parental assignment was performed on a
total of 155 individuals, including 141 off-
spring, 8 candidate mothers, and 6 candidate
fathers. Overall, 98 and 100% of all offspring
were unambiguously allocated to their putative
parental pairs based on the information from
five and seven unlinked loci, respectively. The
cumulative assignment success with real data
for different subsets of the polymorphic loci
was shown in Figure 1. Of the assigned off-
spring, 85% of the genetic mismatches in the
putative parent/offspring analyses occurred at
ucdCg10, ucdCg14, and cmrCg143. The results
of parental pair allocation from PAPA v. 2.0
were in accordance with the two-step method
from CERVUS v. 2.0 with seven loci.
Number of alleles per locus, alleles range,
and expected and observed heterozygosity of
each locus at seven microsatellite loci in brood-
stock and offspring are shown in Table 2. The
number of alleles for all loci in broodstock
ranged from 10 in ucdCg14 to 15 in ucdCg10
and L10, with an average of 13.3 alleles per
locus. There was no allele missing in offspring.
The observed heterozygosity of the offspring
is little higher than that of the parental oys-
ter, but no significant differences were pre-
sented (Wilcoxon matched-pairs signed-rank
test, P = 0.453). However, the expected het-
erozygosity exhibited a significant decrease in
the offspring population (Wilcoxon matched-
pairs signed-rank test, P = 0.016).
Parents Contributions to Reproduction
Parental contribution to reproduction was
compiled as the total number of offspring
assigned to each adult in the above analy-
sis. For males, this number ranged from 8 to
40 offspring, with a mean of 23.5 offspring
per male (variance = 134.3). For females, the
number ranged from 7 to a maximum of 33,
672 LI ET AL.

Table 3. Percentage of parental contribution to reproduction.

Female
Male F#1 F#2 F#3 F#4 F#5 F#6 F#7 F#8 All females

M#1 2.84 3.55 2.13 0.71 1.42 2.13 5.67 2.84 21.28
M#2 1.42 2.13 2.13 2.13 0.00 0.71 0.00 0.71 9.22
M#3 2.84 3.55 1.42 0.71 2.13 2.13 3.55 0.71 17.02
M#4 0.71 2.84 4.26 2.13 0.71 2.13 4.26 1.42 18.44
M#5 2.13 6.38 2.13 5.67 0.00 2.13 7.09 2.84 28.37
M#6 0.00 1.42 0.00 0.00 0.71 0.71 2.84 0.00 5.67
All males 9.93 19.86 12.06 11.35 4.96 9.93 23.40 8.51

with a mean of 17.63 offspring per female
(variance = 73.98). The percentage of offspring
related to each parent contributing to reproduc-
tion was shown in Table 3. Offspring of all par-
ents were observed. The average percentage of
offspring contribution was 12.5% (SE = 0.06)
for females and 16.7% (SE = 0.08) for males.
Chi-square analyses showed significant differ-
ences of reproductive success both in male
(χ 2 = 28.57, P = 0.00) and in female (χ 2 =
27.55, P = 0.00).
Multiple paternity was detected in all eight
female parents. Four to six genotypes of males
were observed in a single clutch of eggs. The
average proportion of offspring sired by differ-
ent males ranking from the most to the least
successful, and the genetically effective pater-
nity frequencies for all the clutches are shown
in Table 4. The average number of fathers con-
tributing to a clutch was 5.13 (SE = 0.64) and
the mean genetically effective paternity fre-
quencies was 4.16 (SE = 0.74).
Multiple maternity was also detected in all
six male parents. Four to eight genotypes of
females were observed in a single clutch. The
average proportion of offspring contributed by
different females in a single clutch ranking
from the most to the least successful, and the
genetically effective maternity frequencies for
the mass spawning are shown in Table 5. The
average number of mothers contributing to a
clutch was 6.83 (SE = 1.60), and the mean
genetically effective maternity frequencies was
5.38 (SE = 1.30).
Effective Population Size
The effective population size calculated for
unequal sex ratio of this mass spawning event
was 13.71, which diminished 2.04% of the
absolute parents. When combined with vari-
ance in reproductive success of individuals, the
calculated Ne was 11.42, which accounted for
another reduction of 16.39% of the absolute
parents. Because all the broodstock participated
in the spawning, the decrease in the value of Ne
was mainly caused by the variance in parental
contributions and unequal sex ratio, of which
the former responded a larger part. The effec-
tive number of males and females are 4.96 and
6.72, respectively. Although all the males and

Table 4. Means ± SD paternity proportions for clutches in which from four to six males were detected to contribute
sperm.

Paternity Paternity Paternity Paternity Paternity

proportion proportion proportion proportion proportion
No. of males No. of of most of second-most of third-most of fourth-most of the least Genetically
participating clutches successful successful successful successful successful effective
in clutches (% of total) male male male male male paternity

4 1 (12.5) 0.43 ± 0.00 0.29 ± 0.00 0.28 ± 0.00 — — 3.27 ± 0.00

5 5 (62.5) 0.48 ± 0.09 0.31 ± 0.03 0.12 ± 0.05 0.11 ± 0.06 0.12 ± 0.00 3.99 ± 0.59
6 2 (25) 0.59 ± 0.38 0.25 ± 0.08 0.14 ± 0.00 0.10 ± 0.00 0.07 ± 0.00 5.03 ± 0.18
 PARENTAGE AND EFFECTIVE POPULATION SIZE ESTIMATION IN OYSTER 673

females were contributing to the next genera-

0.00
0.00
0.00
0.23
Genetically

maternity
effective
tion, the variance of male contribution (134.3)

±
±
±
±
and female contribution (73.98) were great,

3.00
5.12
5.56
6.10
which also indicated that male contribution was
more skewed than that of female. The rate of

successful female
proportion of inbreeding within the spawning (F ) was esti-

0.04 ± 0.01
Maternity

the least mated at a high value of 16.5% per generation.

—
—
—
Discussion
Parentage Analysis
successful female

Recently, the potential of microsatellite loci

proportion of

0.23 ± 0.00
0.06 ± 0.02
fifth-most
Maternity

for pedigree analysis have been studied in

—
—

many aquaculture species, and it has been

shown that the pedigree of mixed populations
could be determined through the use of four
to six microsatellite markers (O’Reilly et al.
successful female
Table 5. Means ± SD maternity proportions for clutches in which from four to eight females were detected to dam.

1998; Perez-Enriquez et al. 1999; Waldbieser

proportion of

0.10 ± 0.00
0.12 ± 0.07
fourth-most
Maternity

and Wolters 1999; Jerry et al. 2004; Dong et al.

—
—

2006; Hatanaka et al. 2006). This can provide

an efficient and economical methodology in
selection programs that will enable the maxi-
mization of genetic response for traits such as
successful female

growth, whereas minimize the potential detri-

proportion of

0.00
0.00
0.00
0.11
third-most
Maternity

mental effects of accumulated inbreeding.

±
±
±
±

Simulation assignment approaches are essen-

0.25
0.15
0.20
0.25

tial to test the theoretical power of these

microsatellite markers and allow the adjustment
of marker-selection toward the best cost/benefit
successful female
of second-most

direction. In this study, we have evaluated

0.00
0.00
0.00
0.04
proportion
Maternity

the performance of seven microsatellite loci

±
±
±
±

reported previously for parentage assessment in

0.25
0.15
0.22
0.16

14 commercial Pacific oyster broodstock and in

141 progenies obtained by mass spawning. The
parentage assignment of both simulated and
most successful
proportion of

real offspring was efficient. The power of these

0.70 ± 0.00
0.25 ± 0.00
0.38 ± 0.10
0.5 ± 0.00
Maternity

female

markers to resolve parentage was generally in

accordance with that predicted by simulation
(Fig. 1).
Although usually low, frequencies of null
alleles above 20% have been reported in oys-
(% of total)
clutches

(16.7)
(16.7)
(16.7)
(50.0)
No. of

ter stocks (McGoldrick et al. 2000). The pres-

ence of null alleles is a classical source of
1
1
1
3

incompatibilities in parentage assignment with

microsatellite loci, with the frequencies above
No. of females

5% are considered to compromise pedigree

participating
in clutches

determination (Marshall et al. 1998). Of the

assigned offspring in this experiment, 85%
of the genetic mismatches were detected in
4
6
7
8
674
three loci (ucdCg10, ucdCg14, and cmrCg143 ),
which were proved by MICRO-CHECKER
analyses to have null alleles. Nonetheless, it
seems that adding these loci in the parentage
analysis increased detection probability and did
not lower the overall confidence level at which
offspring were assigned. Generally, in this study
a higher allocation efficiency of Pacific oyster
parental assignment was attained with six loci
to overcome the assignment failures that may
arise from genotyping errors, non-amplifying or
null alleles. This achievement appears plausi-
ble, given our a priori assignment expectations
based on simulation procedures and on the pre-
dictions of the parentage model of Marshall
et al. (1998).
Genetic Variability, Effective Population Size,
and Parental Contributions
Many theoretical and empirical studies have
provided supporting evidence for the positive
relationship between genetic variation and fit-
ness in animals and plants (Fisher 1930; Allen-
dorf and Leary 1986; Ledig 1986). Allelic
diversity and heterozygosity are both measures
of genetic variation. In this study, there were no
differences of allelic diversity or observed het-
erozygosity (Ho ) between parents and offspring
populations, but the expected heterozygosity
(He ) had a significant decrease in offspring. The
plans of careful selection of ripe broodstock and
scientific induced-spawning method used here
ensured that all parental oysters contributed to
the next generation and to some extent avoided
the missing of alleles and sustained the stability
of observed heterozygosity.
Although it was restricted to analyze the
genetic differences through the traditional mea-
sures of genetic variation, the estimation of
effective population size provides important
information about the genetic change in closed
broodstock populations and hence got hold
of comprehensive attention. Low Ne /N ratios
were reported in many marine organisms
(Gaffney et al. 1992; Hedgecock 1994; Fran-
kham 1995). This could be the result of
high variance in parental contributions, which
decrease genetically the effective size of a
LI ET AL.
population without affecting census population
size. This factor is expected to be especially
important in highly fecund species, in which
most of the mortality occurs during the egg
and larval stages (Hedgecock 1994). Boudry
et al. (2002) showed a high variance in parental
contributions (even when gametic contribu-
tions are balanced) of the Pacific oyster in
a controlled experiment. Sekino et al. (2003)
reported an extreme case for the broodstock
males of Japanese flounder: more than 99% of
the offspring turned out to have been sired by
a single male. The Pacific oyster mass spawn-
ing study reported here seems to be a compar-
atively balanced case that all the broodstock
contributed to the reproduction; this is proba-
bly because of the mass spawning method and
careful selection of ripe individuals used here.
These strategies can neglect some factors such
as non-random mating and poor gamete quality
that will result in uneven parental contribution
and successive decline of effective population
size. However, because of the high mortality
during metamorphism, it may not be so bal-
anced for the adult data for this species. Even
with all broodstock participating, obvious dif-
ferences between parental contributions existed
in the study. Chi-square analyses showed sig-
nificant differences of reproductive success both
in male (χ 2 = 28.57, P = 0) and female (χ 2 =
27.55, P = 0). The high variance in parental
contributions may be influenced by many fac-
tors such as differential viability among geno-
types, uneven sex ratios, and fluctuating family
sizes (Hedgecock and Sly 1990; Gaffney et al.
1992; Boudry et al. 2002; Beaumont and Hoare
2003).
Variance in the reproductive success of males
is a striking feature of many animal species, and
this was proposed as a common limitation to
Ne in marine mass spawning species (Hedge-
cock 1994). Male reproductive competition for
fertilization has been documented in a rapidly
increasing number of genetic paternity stud-
ies of a wide range of fish species (DeWoody
and Avise 2001). Some authors suggest the dif-
ferences between male’s reproductive success
that has been documented in fish are largely
the result of male size rank, with larger males
 PARENTAGE AND EFFECTIVE POPULATION SIZE ESTIMATION IN OYSTER
acquiring a larger proportion of offspring than
smaller males (Brawn 1961; Hutchings et al.
1999; Bekkevold et al. 2002); others show-
ing a correlation between male condition fac-
tor/weight and reproductive success with males
of higher condition factor/weight siring a large
proportion of offspring (Fessehaye et al. 2006);
also sperm competition play an important role
in reproductive success (Stockley et al. 1997).
Our paternity analyses revealed that multiple
paternity appeared in all clutches, and hence
that oyster males experience high sperm com-
petition intensity as the effective paternity fre-
quency per clutch was significantly > 1, which
was also the truth in maternity analyses, sug-
gesting additional mate “choice” taking place
at the level of gametes as reported for Atlantic
cod (Rakitin et al. 1999).
Avoiding inbreeding in shellfish is essential
for efficient long-term breeding strategies. In
population under selection, inbreeding produces
negative effects such as increased homozygos-
ity, which leads to the increased chance of
expression of lethal or lower fitness recessive
genes, inbreeding depression, and reduction of
genetic variance (Falconer 1989). Evans et al.
(2004) proved that the performance traits of
Pacific oysters are significantly affected by rel-
atively low levels of inbreeding. Inbreeding
depression is also observed in the traits of
deformity rate and survival in the first gener-
ation of a full-sib family of the Pacific abalone
(Park et al. 2006). The long-tem rate of inbreed-
ing is determined not only by a planned mat-
ing system but also by an effective popula-
tion size for populations under artificial selec-
tion (Wray and Thompson 1990). Assuming no
selection and random mating, the coefficient of
inbreeding estimated from effective population
size per generation was 4.4% for this study
(Falconer 1989). Bijma (2000) suggested an
inbreeding constraint of 1% per generation for
breeding programs. However, the mass spawn-
ing breeding of Pacific oyster in this study had
a much higher F values of 16.5%, suggesting
a significant inbreeding depression might occur
in this small-scale, mass spawning study. In
order to reduce the risks of inbreeding, partic-
ularly within an unrecorded selective breeding
675
hatchery population, the Ne per generation in
a hatchery population should be increased by
using methods not only adding the number of
parents but also equalizing the sex ratio and
contribution of each parent for future oyster cul-
tivation generations.
Conclusions
The high accuracy and economical method-
ology of microsatellite parentage determina-
tion exhibited in this study proved that the
microsatellite-based pedigree tracing is an effe-
ctive way for monitoring genetic changes in
reproductive programs. The effective popula-
tion size estimated from this pilot experimenta-
tion diminished to 11.42, timely reflecting the
change in genetic variability. The large vari-
ance in parental contributions and unequal sex
ratio were considered the main limitation to Ne
in this mass spawning event. This may give
us an insight into the Pacific oyster cultiva-
tion in present China that in order to avoid the
deleterious effects of inbreeding depression and
maximize the genetic gains of Pacific oyster
cultivation in the long run, not only increas-
ing the number of parents, but also equalizing
the sex ratio and their contribution should be
considered to increase the effective number of
breeders.
Acknowledgments
The authors would like to thank the Haili
Hatchery of Haiyang City for its support in
the breeding experiment. The study was sup-
ported by the grants from National High Tech-
nology Research and Development Program
(2006AA10A409, 2007AA09Z433) and Chi-
nese Ministry of Education (No. 707041).
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