Parentage assignment in hybrid abalones (Haliotis rufescens 3 Haliotis discus hannai) based on...

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



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.



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.



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



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).

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