Parentage assignment in hybrid abalones (Haliotis
rufescens 3 Haliotis discus hannai) based on
microsatellite DNA markers
Fabiola Lafarga-de la Cruz, Andrea Aguilar-Espinoza & Cristian Gallardo-Esc�arate
Laboratory of Biotechnology and Aquatic Genomics Interdisciplinary, Center for Aquaculture Research (INCAR),
University of Concepci�on, Concepci�on, Chile
Correspondence: C Gallardo-Esc�arate, Laboratory of Biotechnology and Aquatic Genomics Interdisciplinary, Center for Aquacul-
ture Research (INCAR), University of Concepci�on, PO Box 160-C, Concepci�on, Chile. E-mail: [email protected]
Abstract
Parentage analysis in aquaculture determines
genealogical relationships between broodstock and
progeny when the parents are unknown. Thus,
parentage analysis is a useful tool to establish ped-
igree reports in molecular-assisted selection pro-
grams. Here, we evaluated 10 heterologous
microsatellite markers for parentage assignment in
abalone hybrids produced from 43 abalone brood-
stocks of red abalone (Haliotis rufescens) and Japa-
nese abalone (H. discus hannai). The allele
frequencies, exclusion probabilities and broodstock
contributions were calculated using CERVUS,
PAPA and GERUD software. The polymorphic
information content (PIC) values showed that most
of the microsatellite loci were highly informative
(>0.7) and more than 90% of parentage assign-
ment was possible with a minimum of 5–6 micro-
satellite markers. Parentage assignment for hybrid
and pure-red progeny showed a better perfor-
mance than pure-Japanese progeny. This result
could be due to the high level of allele loss in the
parental genotypes. In addition, results indicated
that only two sires contributed over 80% and 90%
of red and hybrid progenies, respectively. This
study gives a new molecular tool to support mar-
ker-assisted selection in abalone hybrids produced
in Chile.
Keywords: hybrid abalone, Haliotis rufescens,
Haliotis discus hannai, microsatellite markers,
parentage assignment
Introduction
The family Haliotidae is represented by more than
65 species, of which some are commercially
important for fisheries or aquaculture (Geiger &
Poppe 2000). Over the last decade, abalone aqua-
culture has become widespread worldwide in
response to the over-exploitation of most wild fish-
eries (Gordon & Cook 2004). Abalone aquaculture
in Chile is currently based on two introduced spe-
cies, the red abalone, Haliotis rufescens (Swainson)
and the Japanese abalone, H. discus hannai (Ino),
both introduced in the 1980s. Thereafter, the
abalone industry has grown rapidly, reaching pro-
duction levels of 647 TM (US$ 18.5 million),
establishing Chile as the fifth largest abalone sup-
plier in the world. Red abalone production
accounts for 98% of total production because the
species has adapted better to local conditions than
has the Japanese abalone (Flores-Aguilar,
Guti�errez, Ellwanger & Searcy-Bernal 2007). As a
consequence, abalone hybrids between these spe-
cies have been developed to diversify and improve
Chilean aquaculture production. Abalone hybrids
have shown better performance under commercial
conditions in terms of growth and survival rates
and thermal tolerance in comparison to their
parental species [see details in (Lafarga de la Cruz
& Gallardo-Esc�arate 2011)].
Although red and Japanese abalone have been
cultivated for almost 30 years in Chile, genetic
studies of culture populations are scarce (Lafarga-
de la Cruz, Aguilera-Mu~noz, Del R�ıo-Portilla &
© 2013 Blackwell Publishing Ltd 1
Aquaculture Research, 2013, 1–10 doi:10.1111/are.12169
Gallardo-Esc�arate 2010). Loss of genetic variability
in aquaculture species can increase with lack of
genetically controlled breeding programs and
unknown pedigree information (Hara & Sekino
2007; Johnson, Rexroad, Hallerman, Vallejo & Palti
2007; Sonesson 2007). Thus, breeding strategies
assisted by molecular DNA markers could be
improved with detailed knowledge of the genetic
composition of broodstock and offspring, minimiz-
ing inbreeding effects (Perez-Enriquez, Takagi &
Taniguchi 1999; Kim, Morishima, Satoh, Fujioka,
Saito & Arai 2007). Selective breeding programs
have been successfully applied to rainbow trout
(Henryon, Berg, Olesen, Kjaer, Slierendrecht,
Jokumsen & Lund 2005), common carp (Vande-
putte 2003), sea bass (Garcia de Leon, Cannone,
Quillet, Bonhomme & Chatain 1998; Costa, Vande-
putte, Antonucci, Boglione, Menesatti, Cenadelli,
Parati, Chavanne & Chatain 2010), Pacific thread-
fin (Wang, Iwai, Zhao, Lee & Yang 2010), oysters
(Lallias, Boudry, Lap�egue, King & Beaumont
2010), mussels (MacAvoy, Wood & Gardner 2008)
and abalones (Kijima, Li & Park 2002; Vandeputte,
Mauger & Dupont-Nivet 2006; Hayes, Baranski,
Goddard & Robinson 2007; Kube, Appleyard & Elli-
ott 2007; Slabbert, Bester & D’Amato 2009).
The ability to identify individual genotypes in
culture systems can solve problems associated with
traditional practices of selective breeding programs
where individual family lines are separately culti-
vated. Rearing offspring from multiple parents
together permits a better evaluation of genetic
effects on a targeted trait by reducing the con-
founding effects of environment (Herbinger, O’Reil-
ly, Doyle, Wright & O’Flynn 1999). In this study,
43 putative parents were used to produce two
control crosses (pure lines) and one hybrid cross
between red abalone (♀) and Japanese abalone
(♂). To perform a parentage analysis, 10 microsat-
ellite loci were genotyped and the minimum num-
ber of loci to obtain a high percentage of
parentage assignment was evaluated. Further-
more, the relative contribution of each broodstock
to offspring was estimated.
Materials and methods
Offspring production
Red abalone H. rufescens (n = 22) and Japanese
abalone H. discus hannai (n = 21) broodstock were
individually tagged and maintained in separate
tanks (by species and sex) in a flow-through sea-
water system and fed twice a week with a mix diet
of brown algae (Macrocystis sp., Lessonia sp.) and
red algae (Gracilaria sp.). The mean temperature
for broodstock was of 15°C (�1°C) and 17°C(�1°C) for red and Japanese abalone respectively.
Before spawning, male and female of each spe-
cies were put separately into tanks of 8 L with
1-lm filtered seawater (FSW). Spawning was artifi-
cially induced through chemical and ultraviolet
(UV) irradiated seawater stimulation, as described
by Morse, Duncan, Hooker & Morse (1976), with
some modifications in the duration and doses of
induction. Three experimental crosses were estab-
lished, each consisting of different gamete combi-
nations, as follows: to produce pure-red offspring,
15 red dam gametes were pooled and fertilized
with 7 red sire gametes. To produce pure-Japanese
offspring, 15 pooled Japanese dam gametes were
fertilized with 6 pooled Japanese sire gametes.
Finally, the same dams used for pure-red offspring
(15) and sires used for pure-Japanese offspring (6)
were used to produce abalone hybrid offspring
(H. rufescens 9 H. discus hannai). Fertilization and
settlement and mortality rates were recorded for
each abalone cross.
DNA extraction and PCR amplification
Epipodial tissue was taken from all tagged putative
broodstock (n = 43) and stored in 100% ethanol,
while whole juveniles (n = 50 for each cross) were
stored in 100% ethanol for DNA analysis. DNA
was extracted using the EZNA kit (Omega Bio-Tek,
Norcross, GA, USA) in accordance with the manu-
facturer’s instructions. DNA quantity and purity
were measured with a ND1000 spectrophotometer
(NanoDrop� Technologies, Wilmington, DE, USA)
and quality was tested in 1% agarose gel electro-
phoresis.
Microsatellite loci used were previously isolated
for H. kamtschatkana (Hka3, Hka28, Hka40, Hka56
and Hka80) (Miller, Laberee, Kaukinen, Li & With-
ler 2001), H. discus hannai (Awb026, Awb033,
Awb041 and Awb062) (Sekino, Saido, Fujita,
Kobayashi & Takami 2005) and H. corrugata
(Hco97) (D�ıaz-Viloria, P�erez-Enr�ıquez, Fiore-Ama-
ral, Burton & Cruz 2008). PCR amplification was
carried out in a final volume of 15 lL containing
1X PCR buffer, 0.2 lg lL�1 BSA, 0.2 mM dNTPs
mix, 1.5 mM MgCl2, 0.5 lM of each primer set
(forward primers were 5′ end-labelled with FAM or
© 2013 Blackwell Publishing Ltd, Aquaculture Research, 1–102
Parental assignment in hybrid abalone based on SSR F Lafarga-de la Cruz et al. Aquaculture Research, 2013, 1–10
HEX dyes), 1.25 U KAPA Taq DNA Polymerase
(Kapa Biosystems�, Middlesex Country, MA, USA)
and about 10 ng template DNA. PCR was per-
formed with a VeritiTM thermocycler (Applied Bio-
systems�, Foster City, CA, USA), as described by
Lafarga-de la Cruz, Aguilera-Mu~noz, et al. (2010).
To evaluate the amplifications, amplicons were
first run through 1.8% agarose gel electrophoresis.
Later, PCR products were mixed up to three,
depending on their allelic size range and fluorescent
dyes used. Microsatellite fragment analysis was per-
formed in an ABI3730XL automated DNA sequen-
cer (Applied Biosystem�). Alleles were assigned
according to their relative sizes estimated with
molecular size marker GeneScanTM 400HD ROXT-
M(Applied Biosystems�) using GeneMarker v1.75
software (Softgenetics�, State College, PA, USA).
Parentage and broodstock contribution analysis
Possible genotyping errors associated with micro-
satellite analysis, such as stutter bands, null alleles
and large allele dropout were tested with MICRO-
CHEKER v2.3.3 software (Oosterhout, Hutchinson,
Wills & Shipley 2004). The utility of microsatellite
loci to determine parentage in the three experimen-
tal crosses was assessed using a likelihood-based
method with CERVUS v3.0 (Marshall, Slate, Kruuk
& Pemberton 1998; Kalinowski, Taper & Marshall
2007). This software is designed for
co-dominant molecular markers and calculates
allelic frequencies, expected and observed hetero-
zygosity and polymorphic information content
(PIC) for each locus. PIC is a measure of informa-
tiveness for co-dominant molecular markers related
to expected heterozygosity, assuming Hardy–Wein-
berg equilibrium (Botstein, White, Skolnick & Davis
1980). On the other hand, parentage exclusion
probabilities (PE) for the first (EXC1) and second
(EXC2) parent of each locus for each cross were cal-
culated using GERUD v2.0 software (Jones 2005).
The PE for all microsatellite loci for EXC1 (first par-
ent) is defined as the combined power of the set of
loci that allows the exclusion of a candidate parent
when neither parent is known (Marshall et al.
1998). On the other hand, PE for all microsatellite
loci for EXC2 (second parent) is defined as the com-
bined power of the set of loci that allows the exclu-
sion of a candidate parent when one parent is
known with certainty and the other is unknown.
Parental assignment percentages and broodstock
contributions were calculated with CERVUS v3.0
and PAPA v2.0 (Duchesne, Godbout & Bernatchez
2002) software. CERVUS v3.0 calculates likelihood
ratios for paternity inference and defines a statisti-
cal delta. The delta assessed the reliability of par-
entage assignment to the most likely candidate
parent, and it was defined as the difference in LOD
scores between the most likely and the second
most likely candidate parent. The candidate parent
with the highest (most positive) LOD score is the
most likely candidate (Dong, Kong, Zhang, Meng
& Wang 2006) and a LOD score of 3 is considered
the critical value above which assignment can be
accepted with 95% confidence (Slate, Marshall &
Pemberton 2000). The parameters for parentage
assignment used here were: 10 000 replication
cycles, a strict confidence level for allocations of
95% and a relaxed level of 80%, and a typing
error rate of 1%. Likewise, PAPA v2.0 is also a
parental pair allocation and prediction program.
However, it uses likelihood scores to allocate
parental pairs. Probabilities for each offspring are
calculated against each potential parent pair. The
parent pair with the highest likelihood is the
assigned parentage. Offspring are not allocated
when no parents show any likelihood or when
two or more parental pairs share the same likeli-
hood. As well, a degree of transmission error due
to allele mistyping and/or genetic mutation can be
fixed in PAPA v2.0 software. This transmission
error rate can be uniform (all errors assumed to be
equally likely), non-uniform (to reflect greater
misscoring among alleles of similar mobility) or
simply in the transmission error rate (Herlin, Tag-
gart, McAncrew & Penman 2007). In this study,
we used a uniform transmission error rate of 0.02.
Results
Broodstock genotypes
The fertilization rates were 90%, 85% and 60% for
each cross, respectively, while settlement rates
were 12.3%, 18.6% and 16.7%. The mortality rate
was similar among the experimental trials, with a
normal mean value of 48.4 � 2.1%. Regarding
broodstock genotypes, the mean PIC values were
0.741 for the red abalone cross, 0.606 for the
Japanese cross and 0.748 for the hybrid cross
(Table 1). Figure 1 shows the PE calculated for all
polymorphic microsatellite loci assessed for EXC1
(first parent) and EXC2 (second parent) for each
cross. For EXC1, locus Hka80 contributed 69% of
© 2013 Blackwell Publishing Ltd, Aquaculture Research, 1–10 3
Aquaculture Research, 2013, 1–10 Parental assignment in hybrid abalone based on SSR F Lafarga-de la Cruz et al.
allocation for the red abalone cross, locus Hka56
contributed 37% of assignment for the Japanese
abalone cross and locus Hco97 contributed 65% of
assignment for the hybrid cross. For EXC2, locus
Hka80 provided 81% of assignment for red aba-
lone cross, locus Hka56 provided 55% for the Japa-
nese cross and locus Hco97 provided 79% of
assignment in the hybrid cross. For the red and
hybrid abalone crosses, the high probability values
indicate that these loci would be effective in par-
entage assignment. In contrast, the parentage
exclusion probability values for each locus for the
Japanese abalone cross were lower (Table 1).
Parentage assignments
For red abalones produced by 15 dams and 7
sires, 100% parentage assignment of pure-red
Table 1 Genetic diversity estimates [Number of alleles
(k), observed heterozygosity (Ho), expected heterozygosity
(He), polymorphic information content (PIC), probabilities
of exclusion based either on the genotype of no parent
known (EXC1) or one parent known (EXC2) and null
frequency] for the 10 microsatellites loci analysed at the
abalone broodstock and their hybrids
Locus
Genetic
variability
indices
Crosses
Red
abalone
Japanese
abalone Hybrid
Hka3 k 14 3 12
Ho 0.868 0.906 0.961
He 0.888 0.633 0.847
PIC 0.868 0.547 0.818
EXC1 0.609 0.196 0.510
EXC2 0.758 0.334 0.679
Null
frequency
0.0033 �0.1897 �0.0725
Hka28 k 10 5 10
Ho 0.722 0.489 0.588
He 0.855 0.657 0.834
PIC 0.829 0.584 0.803
EXC1 0.526 0.228 0.484
EXC2 0.692 0.381 0.655
Null
frequency
+0.0699 +0.1510 +0.1653
Hka40 k 12 5 14
Ho 0.849 0.615 0.820
He 0.866 0.609 0.874
PIC 0.843 0.536 0.852
EXC1 0.557 0.194 0.580
EXC2 0.718 0.339 0.735
Null
frequency
+0.0037 �0.0041 +0.0230
Hka56 k 15 10 13
Ho 0.509 0.438 0.580
He 0.905 0.766 0.861
PIC 0.887 0.723 0.838
EXC1 0.653 0.370 0.553
EXC2 0.790 0.548 0.714
Null
frequency
+0.279 +0.2737 +0.1904
Hka80 k 15
Ho 0.566
He 0.917
PIC 0.901
EXC1 0.687
EXC2 0.815
Null
frequency
+0.2318
Awb026 k 7 5 8
Ho 0.463 0.404 0.698
He 0.422 0.556 0.630
PIC 0.396 0.489 0.555
EXC1 0.095 0.158 0.215
EXC2 0.244 0.299 0.360
Null
frequency
�0.059 +0.1339 �0.0600
Table 1 (continued)
Locus
Genetic
variability
indices
Crosses
Red
abalone
Japanese
abalone Hybrid
Awb033 k 10 7 9
Ho 0.788 0.667 0.333
He 0.810 0.704 0.740
PIC 0.775 0.655 0.702
EXC1 0.475 0.290 0.346
EXC2 0.649 0.466 0.528
Null
frequency
�0.0182 +0.0100 +0.4011
Awb041 k 3 5 5
Ho 0.321 0.604 0.412
He 0.318 0.755 0.747
PIC 0.280 0.706 0.697
EXC1 0.050 0.339 0.331
EXC2 0.149 0.516 0.507
Null
frequency
+0.0362 +0.1036 +0.2984
Awb062 k 10 7 9
Ho 0.788 0.780 0.412
He 0.810 0.686 0.609
PIC 0.775 0.637 0.581
EXC1 0.440 0.273 0.224
EXC2 0.617 0.448 0.411
Null
frequency
�0.0021 �0.0743 +0.2320
Hco97 k 14 3 14
Ho 0.537 0.415 0.750
He 0.857 0.661 0.905
PIC 0.836 0.580 0.887
EXC1 0.552 0.214 0.652
EXC2 0.714 0.360 0.790
Null
frequency
+0.2181 +0.227 +0.0820
© 2013 Blackwell Publishing Ltd, Aquaculture Research, 1–104
Parental assignment in hybrid abalone based on SSR F Lafarga-de la Cruz et al. Aquaculture Research, 2013, 1–10
offspring was achieved through 8 microsatellite
loci (Fig. 2a). In Japanese abalones produced by
15 dams and 6 sires, 100% of the pure-Japanese
offspring was achieved with 9 microsatellite loci
fixed at an 80% relaxed confidence level. However,
with a strict 95% confidence level, it was not
possible to assign offspring correctly (Fig. 2b). In
hybrid abalones produced using 15 dams from red
abalone and 6 sires from Japanese abalone, 100%
parentage assignment of hybrid offspring was
achieved by alleles matching of 8 microsatellite
loci (Fig. 2c).
Broodstock contribution
The parentage analysis established the contribu-
tion of males and females to the offspring of red
abalone and hybrid abalone crosses. In the case of
(a)
(b)
(c)
Red abalones
Japanese abalones
Hybrid abalones
Figure 1 Combined probabilities
of exclusion calculated overall
microsatellite loci analysed for
EXC1 and EXC2 for abalone
crosses.
© 2013 Blackwell Publishing Ltd, Aquaculture Research, 1–10 5
Aquaculture Research, 2013, 1–10 Parental assignment in hybrid abalone based on SSR F Lafarga-de la Cruz et al.
the Japanese abalone cross, the great number of
allele loss in the parental genotype matrix data
made it impossible to accurately establish contri-
butions to pure-Japanese offspring. Table 2 sum-
marizes broodstock contributions. For the red
abalone cross, 59.4% of pure-red offspring were
derived from sire 3 and 21.9% by sire 4, while
maternal contributions were attributed as follows:
25% to dam 8, 15.6% to dam 2, 15.6% to dam
13 and 12.5% to dam 1, accounting for a total
68.6% of the offspring. In the hybrid abalone
cross, 56.3% of hybrid progeny were derived from
sire 1 and 37.5% from sire 4; while 21.9% of the
juvenile seed surveyed were derived from dam 5,
21.9% from dam 6, 18.9% from dam 14 and
12.5% from dam 2, accounting for approximately
75%.
Discussion
Microsatellite DNA markers for parentage
assignment
Sustainable aquaculture of H. rufescens, H. discus
hannai and their inter-specific hybrid require basic
genetic knowledge of the stock structure to estab-
lish appropriate broodstock management and effec-
tive selective breeding programs. The first phase to
establish a molecular-assisted selection program
(M.A.S) is to determinate the parental and pedigree
(a)
(b)
(c)
Red abalones
Japanese abalones
Hybrid abalones
Figure 2 Cumulative assignment
success of progeny to correct dam,
sire and parent pair, respectively,
based on strict (95%) and relaxed
(80%) confidence levels of parent
and offspring genotypes from
abalone crosses.
© 2013 Blackwell Publishing Ltd, Aquaculture Research, 1–106
Parental assignment in hybrid abalone based on SSR F Lafarga-de la Cruz et al. Aquaculture Research, 2013, 1–10
information of broodstock to avoid inbreeding
effects. Microsatellite markers are useful DNA
molecular tools to do this given their relatively
high genome abundance, high mutation rate and
the high degree of polymorphism, as well as the
co-dominant Mendelian nature of inheritance (Liu
& Cordes 2004). The level of heterozygosity and
polymorphic informative content of each microsat-
ellite locus determines its ability as a molecular
parentage assignment tool.
In this study, we used a set of microsatellite loci
previously isolated for H. discus hannai and another
set isolated for H. kamtschatkana and H. corrugata,
which have shown successful cross-amplification
in our studied target species (Lafarga-de la Cruz,
Amar-Basulto, R�ıo-Portilla & Gallardo-Esc�arate
2010). Earlier research on the genetic variability
in cultured populations revealed that this set of
microsatellite loci presents moderate levels of
observed heterozygosity (Lafarga-de la Cruz, Aguil-
era-Mu~noz, et al. 2010; Lafarga-de la Cruz, Amar-
Basulto, et al. 2010). According to Botstein et al.
(1980), a co-dominant loci is highly informative
for scores of PIC >0.5, reasonably informative for
0.5 > PIC > 0.25 and slightly informative for PIC
<0.25. Therefore, loci with many alleles and a PIC
near 1 are most desirable. In our results, the mean
PIC value was over 0.5 (0.606–0.748), suggesting
that these microsatellite loci could be useful in
parentage assignment for each cross. Another way
to evaluate the usefulness of loci in parentage
analysis is their combined exclusion power values
for two unknown parents (EXC1) and one
unknown parent (EXC2) (Marshall et al. 1998). In
general, our results have shown that the exclusion
power values for the red abalone crosses and
hybrid cross are relatively high, with a total of
eight microsatellite loci that allows allocating
>95% of the progeny. Nevertheless, the exclusion
power values for Japanese cross were quite low
and parentage assignment for this cross could be
not resolved accurately.
Parentage assignment and broodstock
contributions in aquaculture
The capability to assign progeny to a particular
parental pair is contingent upon the number of mi-
crosatellite loci available and their ability to
exclude nonparents, which is strongly correlated
with the allelic diversity of the loci (Garcia de Leon
et al. 1998; Marshall et al. 1998; Selvamani,
Degnan & Degnan 2001). According to Vandeputte
et al. (2006) and Dong et al. (2006), 10–20 vari-
able genetic markers are needed to assign >95% of
individuals to single pairs of parents. However, a
few highly polymorphic loci are sufficient for
successful parentage assignment (Garcia de Leon
et al. 1998; Norris, Bradley & Cunningham 2000;
Selvamani et al. 2001; Dong et al. 2006; Lucas,
Macbeth, Degnan, Knibb & Degnan 2006). From
this study, it is evident that a high number of
microsatellite loci are effective in assigning parents
from offspring of H. rufescens and hybrids of
H. rufescens 9 H. discus hannai. In our case, the
high number of microsatellite loci needed to obtain
>95% progeny assigned can be due to the moder-
ate polymorphism and the heterologous nature of
the microsatellite loci used. Herein, null allele
frequencies among congeneric species rapidly
increase with increased phylogenetic distances
among target species (Hedgecock, Li, Hubert, Buck-
lin & Ribes 2004), producing an under-estimation
of allelic diversity and the combined exclusion
power. In this sense, the presence of null or non-
amplifying alleles (alleles that do not give a PCR
product) in microsatellite loci can affect parentage
Table 2 Percentage of broodstock (Sires and Dams)
contribution for red abalone and hybrid abalone offspring
Cross
Red abalone (%) Hybrid abalone (%)
Sire
1 – 56.3
2 9.4 –
3 59.4
4 21.9 37.5
5 – 6.3
6 9.4 –
7 – –
Dam
1 12.5 6.3
2 15.6 12.5
3 – –
4 6.3 –
5 – 21.9
6 9.4 21.9
7 3.1 –
8 25.0 2.9
9 – –
10 – 3.1
11 – –
12 – –
13 15.6 9.4
14 3.1 18.9
15 3.1 3.1
© 2013 Blackwell Publishing Ltd, Aquaculture Research, 1–10 7
Aquaculture Research, 2013, 1–10 Parental assignment in hybrid abalone based on SSR F Lafarga-de la Cruz et al.
assignment (Dakin & Avise 2004), introducing
substantial errors to empirical assessments by lead-
ing to high frequencies of false parentage exclu-
sion. Furthermore, null alleles could be explained
by the presence of several parental homozygote
genotypes (Hedgecock et al. 2004). This had led to
the suggestion that only heterozygous alleles be
used for assignment tests (Pemberton, Slate,
Bancroft & Barrett 1995).
Broodstock contribution in offspring production
In aquaculture, it is important to assess the repro-
ductive potential of the available broodstock,
which are often collected or introduced from the
wild (Selvamani et al. 2001). Genetic evaluation of
larvae or seeds in abalone aquaculture provides a
realistic estimate of the reproductive potential of
the broodstock. We have demonstrated in this
study that most of the progeny were produced by
only a subset of the potential parents, and specifi-
cally fewer males than females. As observed in off-
spring of the red abalone cross, using pooled
sperm from 7 sires, 81.3% of the individuals come
from only two males (sire 3 and 4). A similar situ-
ation was also evident for offspring of the hybrid
abalone cross, where sires 1 and 4 had fathered
93.8% of the progeny, while most of the females
contributed in similar proportions to offspring
production in both crosses. Similar results have
been demonstrated for direct crosses of H. asinina
and Crassostrea gigas, where only one or two males
contributed with genetic information for their
progenies (Selvamani et al. 2001; Boudry, Collet,
Cornette, Hervouet & Bonhomme 2002). The prev-
alence of sperm contributions of only two males
for the next generation can be attributed to factors
like sperm quality, sperm–egg interaction, relative
genetic compatibility of the gametes and differen-
tial viability among genotypes, although differen-
tial embryonic or larval survival should also be
considered.
Although high parentage assignment percent-
ages were obtained with heterologous microsatel-
lites, it is necessary to develop species-specific
microsatellite DNA markers for red abalone to
avoid null alleles, genotyping errors and loss of
information. However, it is possible to find a grow-
ing numbers of new type specific molecular mark-
ers from Expressed Sequence Tags (ESTs) for red
and Japanese abalone (Li, Shu, Zhao, Liu, Kong &
Zheng 2010; Qi, Liu, Wu & Zhang 2010; De Wit
& Palumbi 2012; Valenzuela-Mu~noz, Bueno-Ibarra
& Gallardo-Esc�arate 2012; Aguilar-Espinoza,
Valderrama-Aravena, Farlora, Lafarga-de La Cruz
& Gallardo-Esc�arate 2013; Valenzuela-Mu~noz,
Araya-Garay & Gallardo-Esc�arate 2013). ESTs offer
the possibility to detect SSRs or SNPs (Single
Nucleotide Polymorphic) within messenger RNA.
This type of molecular markers could also be
directly associated with coding sequences, making
possible to relate them with known genes for gene
function studies (Ellis & Burke 2007; Abdelkrim,
Robertson, Stanton & Gemmell 2009). Due to the
nature of these markers, they could be powerful
tools for parental analysis in H. spp and other
species. One consideration for SNPs is that some
approaches to data analysis are precluded by the
low per-locus levels for this type of analysis (Jones,
Small, Paczolt & Ratterman 2010).
The data obtained here suggest that in cultured
abalone stocks, where common practices include
mass spawning (Hara & Sekino 2007), all genetic
composition of the broodstock may not always be
represented in the offspring, mainly because only a
few males contribute to the next generation. A
more controlled fertilization procedure may be
required to maintain genetic diversity under
culture conditions as a pivotal factor to establish
molecular-assisted selection in hybrid abalone
breeding programs in Chile.
Acknowledgments
This work has been supported by FONDEF
(D09I1065), FONDAP (15110027) CONICYT,
Chile and CONACYT, Mexico (scholarship number
117673/217652).
References
Abdelkrim J., Robertson B., Stanton J.A. & Gemmell N.
(2009) Fast, cost-effective development of species-
specific microsatellite markers by genomic sequencing.
BioTechniques 46, 185–192.
Aguilar-Espinoza A., Valderrama-Aravena N., Farlora R.,
Lafarga-de La Cruz F. & Gallardo-Esc�arate C. (2013)
Development of novel polymorphic EST-SSR markers
in Californian abalone Haliotis rufescens and genetic
analysis in wild and hatchery-bred populations. Aqua-
culture Research. doi: 10.1111/are.12141.
Botstein D., White R.L., Skolnick M. & Davis R.W.
(1980) Construction of genetic linkage map in man
using restriction fragment length polymorphisms.
American Journal of Human Genetics 32, 314–331.
© 2013 Blackwell Publishing Ltd, Aquaculture Research, 1–108
Parental assignment in hybrid abalone based on SSR F Lafarga-de la Cruz et al. Aquaculture Research, 2013, 1–10
Boudry P., Collet B., Cornette F., Hervouet V. & Bonho-
mme F. (2002) High variance in reproductive success
of the Pacific oyster (Crassostrea gigas, Thunberg)
revealed by microsatellite-based parentage analysis of
multifactorial crosses. Aquaculture 204, 283–296.
Costa C., Vandeputte M., Antonucci F., Boglione C.,
Menesatti P., Cenadelli S., Parati K., Chavanne H. &
Chatain B. (2010) Genetic and environmental influ-
ences on shape variation in the European sea bass
(Dicentrarchus labrax). Biological Journal of the Linnean
Society 101, 427–436.
Dakin E.E. & Avise J.C. (2004) Microsatellite null alleles
in parentage analysis. Heredity 93, 504–509.
De Wit P. & Palumbi S.R. (2012) Transcriptome-wide
polymorphisms of red abalone (Haliotis rufescens) reveal
patterns of gene flow and local adaptation. Molecular
Ecology. doi: 10.1111/mec.12081.
D�ıaz-Viloria N., P�erez-Enr�ıquez R., Fiore-Amaral G., Bur-
ton R.S. & Cruz P. (2008) Isolation and cross-amplifi-
cation of microsatellites in pink abalone (Haliotis
corrugata). Molecular Ecology Resources 8, 701–703.
Dong S., Kong J., Zhang T., Meng X. & Wang R. (2006)
Parentage determination of Chinese shrimp (Fennerope-
naeus chinensis) based on microsatellite DNA markers.
Aquaculture 258, 283–288.
Duchesne P., Godbout M.H. & Bernatchez L. (2002)
PAPA (package for the analysis of parental allocation):
a computer program for simulated and real parental
allocation. Molecular Ecology Notes 2, 191–193.
Ellis J.R. & Burke J.M. (2007) EST-SSRs as a resource
for population genetic analyses. Heredity 99, 125–
132.
Flores-Aguilar R., Guti�errez A., Ellwanger A. & Searcy-
Bernal R. (2007) Development and current status of
abalone aquaculture in Chile. Journal of Shellfish
Research 26, 705–711.
Garcia de Leon F.J., Cannone M., Quillet E., Bonhomme
F. & Chatain B. (1998) The application of microsatel-
lite markers to breeding programmes in the sea bass,
Dicentrarchus labrax. Aquaculture 159, 303–316.
Geiger D.L. & Poppe G. (2000) A conchological iconogra-
phy: The family Haliotidae. Conch Books, Hackenheim,
Germany, 135 pp.
Gordon R.H. & Cook P.A. (2004) World abalone fisheries
and aquaculture update: supply and market dynamics.
Journal of Shellfish Research 23, 935–940.
Hara M. & Sekino M. (2007) Parentage testing for hatch-
ery-produced abalone Haliotis discus hannai based on
microsatellite markers: preliminary evaluation of early
growth of selected straing in mixed family farming.
Fisheries Science 73, 831–836.
Hayes B., Baranski M., Goddard M.E. & Robinson N.
(2007) Optimisation of marker assisted selection for
abalone breeding programs. Aquaculture 265, 61–69.
Hedgecock D., Li G., Hubert S., Bucklin K. & Ribes V.
(2004) Widespread null alleles and poor cross-species
amplification of microsatellite DNA loci cloned from
the Pacific oyster, Crassostrea gigas. Journal of Shellfish
Research 23, 379–385.
Henryon M., Berg P., Olesen N.J., Kjaer T.E., Slierendr-
echt W.J., Jokumsen A. & Lund I. (2005) Selective
breeding provides an approach to increase resistance
of rainbow trout (Onchorhynchus mykiss) to the
diseases, enteric redmouth disease, rainbow trout fry
syndrome, and viral haemorrhagic septicaemia. Aqua-
culture 250, 621–636.
Herbinger C.M., O’Reilly P.T., Doyle R.W., Wright J.M. &
O’Flynn F. (1999) Early growth performance in Atlantic
salmon full-sib families reared in single family tanks ver-
sus in mixed family tanks. Aquaculture 173, 105–116.
Herlin M., Taggart J.B., McAncrew B.J. & Penman D.J.
(2007) Parentage allocation in a complex situation: a
large commercial Atlantic cod (Gadus morhua) mass
spawning tank. Aquaculture, 272S1, S195–S203.
Johnson N.A., Rexroad C.E., Hallerman E.M., Vallejo R.L.
& Palti Y. (2007) Development and evaluation of a
new microsatellite multiplex system for parental alloca-
tion and management of rainbow trout (Oncorhynchus
mykiss) broodstocks. Aquaculture 266, 53–62.
Jones A.G. (2005) GERUD 2.0: a computer program for
the reconstruction of parental genotypes from half-sib
progeny arrays with known or unknown parents.
Molecular Ecology Notes 5, 708–711.
Jones A.G., Small C.M., Paczolt K.A. & Ratterman N.L.
(2010) A practical guide to methods of parentage
analysis. Molecular Ecology Resources 10, 6–30.
Kalinowski S.T., Taper M.L. & Marshall T.C. (2007)
Revising how the computer program CERVUS accom-
modates genotyping error increases success in pater-
nity assignment. Molecular Ecology 16, 1099–1106.
Kijima A., Li Q. & Park C. (2002) Development of genet-
ics and breeding in abalone culture. Fisheries Science
68, 730–733.
Kim S.-G., Morishima K., Satoh N., Fujioka T., Saito S. &
Arai K. (2007) Parentage assignment in hatchery pop-
ulation of brown sole Pleuronectes herzensteini by
microsatellite DNA markers. Fisheries Science 73, 1087
–1093.
Kube P.D., Appleyard S.A. & Elliott N.G. (2007) Selective
breeding greenlip abalone (Haliotis laevigata): Prelimin-
ary results and issues. Journal of Shellfish Research 26,
821–824.
Lafarga de la Cruz F. & Gallardo-Esc�arate C. (2011)
Intraspecies and interspecies hybrids in Haliotis: natu-
ral and experimental evidence and its impact on aba-
lone aquaculture. Reviews in Aquaculture 3, 74–99.
Lafarga-de la Cruz F., Aguilera-Mu~noz F., Del R�ıo-Portilla
M.�A. & Gallardo-Esc�arate C. (2010) Genetic variability
of cultured populations of Red abalone Haliotis
rufescens in Chile: an approach based on heterologous
microsatellites. Journal of Shellfish Research 29,
709–715.
© 2013 Blackwell Publishing Ltd, Aquaculture Research, 1–10 9
Aquaculture Research, 2013, 1–10 Parental assignment in hybrid abalone based on SSR F Lafarga-de la Cruz et al.
Lafarga-de la Cruz F., Amar-Basulto G., R�ıo-Portilla M.�A.
& Gallardo-Esc�arate C. (2010) Genetic Analysis of an
Artificially Produced Hybrid Abalone (Haliotis rufes-
cens 9 Haliotis Discus Hannai) in Chile. Journal of Shell-
fish Research 29, 717–724.
Lallias D., Boudry P., Lap�egue S., King J. & Beaumont A.
(2010) Strategies for the retention of high genetic vari-
ability in European flat oyster (Ostrea edulis) restoration
programmes. Conservation Genetics 11, 1899–1910.
Li Q., Shu J., Zhao C., Liu S., Kong L. & Zheng X. (2010)
Characterization of genic microsatellite markers
derived from expressed sequence tags in Pacific aba-
lone (Haliotis discus hannai). Chinese Journal Oceanology
Limnology 28, 46–54.
Liu Z.J. & Cordes J.F. (2004) DNA marker technologies
and their applications in aquaculture genetics. Aquacul-
ture 238, 1–37.
Lucas T., Macbeth M., Degnan S.M., Knibb W. & Degnan
B.M. (2006) Heritability estimates for growth in the
tropical abalone Haliotis asinina using microsatellite to
assign parentage. Aquaculture 259, 146–152.
MacAvoy E.S., Wood A.R. & Gardner J.P. (2008) Devel-
opment and evaluation of microsatellite markers for
identification of individual GreenshellTM mussels
(Perna canaliculus) in a selective breeding programme.
Aquaculture 274, 41–48.
Marshall T.C., Slate J., Kruuk L.E. & Pemberton J.M.
(1998) Statistical confidence for likelihood-based pater-
nity inference in natural populations. Molecular Ecology
7, 639–655.
Miller K.M., Laberee K., Kaukinen K.H., Li S. & Withler
R.E. (2001) Development of microsatellite loci in pinto
abalone (Haliotis kamtschatkana). Molecular Ecology
Notes 1, 315–317.
Morse D.E., Duncan H., Hooker N. & Morse A. (1976)
Hydrogen peroxide induces spawning in mollusks, with
activation of prostaglandin endoperoxide synthetase.
Science 196, 298–300.
Norris A.T., Bradley D.G. & Cunningham E.P. (2000)
Parentage and relatedness determination in farmed
Atlantic salmon (Salmo salar) suing microsatellite
markers. Aquaculture 182, 73–83.
Oosterhout C.V., Hutchinson W.F., Wills D.P. & Shipley
P. (2004) MICRO-CHECKER: software for identifying
and correcting genotyping errors in microsatellite data.
Molecular Ecology Notes 4, 535–538.
Pemberton J.M., Slate J., Bancroft D.R. & Barrett J.A.
(1995) Nonamplifying alleles at microsatellite loci: a
caution for parentage and populations studies. Molecu-
lar Ecology 4, 249–252.
Perez-Enriquez R., Takagi M. & Taniguchi N. (1999)
Genetic variability and pedigree tracing of a hatchery-
reared stock of red sea bream (Pagrus major) used for
stock enhancement, based on microsatellite DNA
markers. Aquaculture 173, 413–423.
Qi H., Liu X., Wu F. & Zhang G. (2010) Development of
gene-targeted SNP markers for genomic mapping in
Pacific abalone Haliotis discus hannai Ino. Molecular
Biology Reports 37, 3779–3784.
Sekino M., Saido T., Fujita T., Kobayashi T. & Takami H.
(2005) Microsatellite DNA markers of Ezo abalone
(Haliotis discus hannai): a preliminary assessment of
natural populations sampled from heavily stocked
areas. Aquaculture 243, 33–47.
Selvamani M.J., Degnan S.M. & Degnan B.M. (2001)
Microsatellite genotyping of individual abalone larvae:
parentage assignment in aquaculture. Marine Biotech-
nology 3, 478–485.
Slabbert R., Bester A.E. & D’Amato M.E. (2009) Analyses
of Genetic Diversity and Parentage Within a South
African Hatchery of the Abalone Haliotis midae Linna-
eus Using Microsatellite Markers. Journal of Shellfish
Research 28, 369–375.
Slate J., Marshall T. & Pemberton J. (2000) A retrospec-
tive assessment of the accuracy of the paternity infer-
ence program cervus. Molecular Ecology 9, 801–808.
Sonesson A.K. (2007) Marker-assisted selection in aqua-
culture breeding programs. Aquaculture 272, S311–
S311.
Valenzuela-Mu~noz V., Bueno-Ibarra M.A. & Gallardo-Esc�a-
rate C. (2012) Characterization of the transcriptomes of
Haliotis rufescens reproductive tissues. Aquaculture
Research. doi: 10.1111/are.12044.
Valenzuela-Mu~noz V., Araya-Garay J.M. & Gallardo-Esc�a-
rate C. (2013) SNP discovery and High Resolution
Melting Analysis from massive transcriptome sequenc-
ing in the California red abalone Haliotis rufescens.
Marine Genomics. doi: 10.1016/j.margen.2012.1012.
1003.
Vandeputte M. (2003) Selective breeding of quantitative
traits in the common carp (Cyprinus carpio): a review.
Aquatic Living Resources 16, 399–407.
Vandeputte M., Mauger S. & Dupont-Nivet M. (2006) An
evaluation of allowing for mismatches as a way to
manage genotyping errors in parentage assignment by
exclusion. Molecular Ecology Notes 6, 265–267.
Wang H., Iwai T., Zhao B., Lee C.-S. & Yang J. (2010)
Identification of Microsatellite DNA Markers for Pacific
Threadfin Parentage Assignment. Journal of the World
Aquaculture Society 41, 640–647.
© 2013 Blackwell Publishing Ltd, Aquaculture Research, 1–1010
Parental assignment in hybrid abalone based on SSR F Lafarga-de la Cruz et al. Aquaculture Research, 2013, 1–10
Top Related