Review of Aquaculture Genetics and Genomics.

Brendan McAndrew,
Genetics and Reproduction Research Group,
Institute of Aquaculture,
University of Stirling,
Stirling FK9 4LA.
Scotland UK.

General introduction
Aquaculture production has continued to grow at an ever increasing rate from less
than 1 million tonnes in the 1950s to nearly 60 million tonnes in 2004 (FAO 2006).
Much of this increase occurring since the mid 1980s with the vast percentage of the
production being from Asia and the Pacific rim, particularly China. The percentages
by volume are China 69.7%, Rest of Asia 21.9% and Rest of the world 8.1% the
respective values of the production are 51.2%, 29.3% and 19.5%. The Rest of the
world percentage includes Western Europe which accounts for 7.7% of the world total
aquaculture production by value. The European sector is dominated by Atlantic
salmon production but, rainbow trout, mussels, oysters and marine species such as
seabass and seabream also now make significant contributions. Despite the great
importance of the present aquaculture production and the hope that its expansion in
the future will help feed the ever growing world population very little of this
production can be said to be coming from genetically managed or improved strains.
Gjedrem (2005) estimated that less than 5% of world aquaculture production comes
from scientifically managed breeding programmes and much of this from salmonids
in northern Europe and north and South America. This leaves a large proportion of the
world’s farmed fish and shellfish under traditional domestication processes. Many of
the new farmed species are still at an early stage of development and still utilise wild
caught fry or broodstock, particularly in Asia. Gjedrem (2005) postulates that despite
the potential for improvements in line with the domestication of terrestrial animals the
high fecundity of aquatic species resulted in the need for small numbers of broodstock
and therefore rapid inbreeding and loss of viability. Farmers had to constantly replace
stocks from the wild to maintain performance. The evidence is that traditional fish
breeding and management continues to seriously degrade the genetic quality of stock
cut off from continuous replacement by wild or managed fish (Pullin and Capilli,
1988; Eknath and Doyle 1990). The FAO State of World Aquaculture report (2006)
says little about these problems and the potential role of genetics to help improve the
sustainability and efficiency of world aquaculture.

Aquaculture Genetics

The early history of fish genetics has recently been reviewed in chapters in books by
Dunham (2004) and Gjedrem (2005) (but also see Benzie, 1998; Gjedrem 2000;
Hulata 2001). The science of aquaculture genetics has grown quickly over the last 30
years alongside the ever expanding aquaculture industry in Europe and North
America. There has been an increase in the number of breeding programmes within
species such as Atlantic salmon and rainbow trout but there is a growing recognition
that genetics can result in immediate gains and should be incorporated at the very
beginning of the domestication process to avoid genetic degradation of these stocks.
This is notable in the level of funding already advanced for genetic management and
improvement and development of genomic tools for new species such as halibut and
Atlantic cod in Canada and Norway.

Aquatic organisms offer many advantages to the geneticist over terrestrial animals.

High fecundity/External fertilisation

• Means that a large number of gametes can be collected and held and fertilised
under controlled hatchery conditions. Typical fecundity for salmonids is 103
eggs/kg , Cyprinids 105 eggs/kg, Oysters 106 eggs/kg per spawning.
• Enables relatively complex breeding designs to be constructed with large
family sizes enabling more accurate estimates of genetic parameters and high
selection pressures. Hybridisation (inter-specific) has been widely used.
• Short or no multiplication stages are needed between breeding nucleus and
commercial production so genetic improvement can be seen by the industry
immediately.
• Gametes relatively cheap so possible to use techniques that induce high
mortality such as chromosome set manipulations or transgenesis but generate
unique and highly valuable genotypes. E.g. Clones, YY males.

Essentially wild organisms

• High levels of genetic variation have been observed in most wild populations
of aquatic organisms. Heterozygosity 6-10% in fish 10-20% in invertebrates.
• Genetic variation is a prerequisite for selective improvement programmes.
Measured heritabilities tend to be medium to high across a wide range of
traits.
• Most species sub-divided into isolated populations that may have genetically
differentiated to adapt to differing environments. Comparisons possible to
identify significantly better founder stocks for aquaculture.
• Few inbred strains available the most developed fish strains are usually
ornamental.

Plastic Phenotypes
• The same genotype can have many possible phenotypes!
• Possible to change sex of many aquatic organisms by temperature or by
administering hormones during sexually labile period of development. This
enables single sex stock to be generated directly or through controlled
breeding.
• It is possible to change the ploidy state and levels of genetic variation by
chromosome set manipulation using temperature or pressure shocks. This
enables sterile triploid fish or unique genotypes such as YY males or isogenic
double haploid clonal lines to be produced.
• Possible to modify the morphology and meristics of scales, fin rays, muscle
precursor cells or other structures by rearing at different temperatures.
• Possible to advance or delay sexual maturation or increase or decrease the age
of onset of traits such as smoltification in salmonids by photoperiod
manipulation.
Some disadvantages.

Despite all these advantages genetic progress has been hindered by the general lack of
knowledge on the biology of many aquatic species. The different spawning
behaviours and the small size of young aquatic organisms make it very difficult to
individually physically mark them at an early stage in their development. In the past
this meant that most improvement programmes used a mass selection approach to
improve obvious phenotypic traits such as scale pattern, colour, growth rate, and body
conformation. Mass selection can result in rapid inbreeding unless measures are taken
to minimise the incidence of related mating. Inbreeding can be minimised through
maintaining a large effective population number (Ne) and through the adoption of line
crossing schemes to delay and reduce the relatedness of any future crosses ( see Tave
1992). When well managed this approach has resulted in improvement (many of the
Eastern European carp programmes came under this heading) but has also in many
circumstances compromised the genetic resource of the species through the loss of
genetic variation caused by genetic drift and inbreeding (the case today in many Asian
carp hatcheries).

In order to undertake the more efficient but complex family selection programmes it
was necessary to use family units so individual families could to be reared in separate
tanks or cages up to a size for physical tagging. Tagging technology has also
improved from the use of external tags or freeze branding that that disfigured fish or
made them more susceptible to disease to the widespread adoption of Passive
Integrated Transponder (PIT) tags that are injected intramuscularly or intra-peritoneal
so avoiding loss. Family units and PIT tagging are now a common feature of salmon
and trout improvement programmes worldwide.

Not all hatchery producers can afford a family unit and not all species can be
individually stripped and their young be reared separately for reasons of biology or
logistics. It was with the recent advances in DNA marker development that it became
possible to individually identify offspring from known genotyped parents making it
possible to reconstruct pedigrees retrospectively after a period of rearing, even in
large communal populations. With this technology broodstock management,
replacement and improvement becomes available to a much greater range of species
and farm based environments. This approach also opens up the possibilities for the
traditional breeding programmes to work with greater numbers of fish in commercial
environments in which tags would be unacceptable, and take advantage of
serendipitous events, e.g. disease outbreaks in untagged populations, regenerate
pedigrees from untagged backup populations in case of losses in breeding nuclei,
compare results from family unit and communally reared fish to assess magnitude of
environmentally induced differences.

It is now possible to overcome the main disadvantages of working with aquatic

organisms and undertake broodstock management, replacement and genetic
improvement using a variety of approaches that can be matched to the biology as well
as the available resources for most farmed aquatic species. The speed of technological
development particularly in genomics has been rapid and its uptake in the aquatic
sciences has become widespread. It is still unclear how much of this will eventually
benefit the aquaculture industry. We have been in a phase of developing the tools
needed to effectively use this technology to inform farmers, breeders, feed companies,
and vaccine manufacturers on how they should integrated the knowledge gained into
their existing procedures. Some of the issues outlined will be expanded in the
following sections.

Development of Genetic Tools

Classical breeding programmes

These have been applied to all of the major animal and plant agricultural species and
have proved highly efficient in improving the yield and quality of the food we eat
today. Man has selected the animals and plants through a domestication process that
has been going on for thousands of years without ever understanding the biological or
genetic processes involved. In many cases it is impossible to assess the degree of
change since many of these ancestral populations no longer exist. Scientifically driven
genetic improvement of terrestrial animals and plants began in the 1930s as the
theoretical basis of quantitative genetics was developed (Lush 1949). Quantitative
genetics was widely adopted supported and funding made available for national and
international institutions aimed at the improvement of terrestrial farm animals. More
recently these activities have been taken over by multinational breeding companies.
This work has resulted in significant improvements in yield particularly over the last
40 years in poultry (meat yield and FCR >300%) Havenstein et al. (2003), dairy cattle
milk yield in 20 years (>100%)( Shook, 2006) and pig meat yield (>200%) in
animals that where nominally already domesticated and genetically improved. This
was seen as being a cost effective way of improving production with cost benefits
ranging between 1:5 to 1:50 depending on the species.

Common carp.
In fish classical selective improvement programmes were established after the second
world war for European common carp in many Central and Eastern European
countries resulting in strains being developed to improve yield from semi-intensive
and intensive pond culture systems (Kirpichnikov 1981). These strains have
subsequently been widely distributed around the world and contribute to the vast bulk
of freshwater farm production in Europe, China and Asia. The history of the
development of some of these carp strains is not well documented. Carp breeding and
testing stations such as that at Szarvas in Hungary maintain landrace strains collected
from around the country as well as improved strains developed from these using mass
selection and crossbreeding (Bakos et al. (2002) also Network of Aquaculture Centers
in Central and Eastern Europe (NACEE). There have been programmes to further
develop these carp in the countries to which they have been imported to better adapt
them to new conditions. Such programmes have been well documented from Israel,
Vietnam and India (Moav and Wohlfarth 1968: Penman et al. 2005 also Network of
Aquaculture Centres in Asia Pacific (NACA)). It is now recognised that many of these
strains no longer respond to selective improvement for growth performance and many
of these programmes utilise some form of multi-strain cross breeding programme.
This is probably because of high selection intensities and the lack of parentage
assignment resulted in a rapid reduction in Ne and loss of variation in many of these
strains. This has to be contrasted with the continued gains in chickens and pigs from
properly managed breeding flocks and herds that have been closed for over 30 years.
The radically different tropical environments into which these carp have been
introduced are causing new production problems not seen in carp within their normal
temperate range such as early or precocious maturation.
Rainbow trout
Rainbow trout has also been the subjected to long-term selective improvement in the
USA initially by Donaldson and Olson (1955) and later more scientifically by Kincaid
et al. (1977), Gall and Huang (1988 a, b) and Gunnes and Gjedrem (1981) and
Gjedrem (1992) for sea grown trout. The largest supplier of eggs to the rainbow trout
industry is Trout lodge and they have only been involved in a family based breeding
programme for the past 5 years. There are breeding programmes in Finland (Kause et
al. 2002, 2003) and Denmark (Henryon et al. 2002). Rainbow trout is another species
that has had a long history of domestication despite this there are few scientifically
managed breeding programmes and defined improved strains. It is difficult to assess
how much of the vast literature on the biology, physiology, endocrinology and
genetics of rainbow trout that has been produced has actually contributed towards the
improvement of rainbow trout as a farmed species. Much of this work has been done
in research institutes and little has been adopted by the industry. Short term
improvements such as all female production or all female triploids have been widely
adopted but the majority of eggs still come from mass selected domesticated strains.

Atlantic salmon
Probably the best documented of the modern scientifically based fish breeding
programmes is that of the Norwegian salmon established in 1971, not long after the
first salmon were being ongrown in pens. The breeding programme was well funded
and benefited from the input of established animal breeders. The breeding programme
assessed the performance of over 40 individual families from 40+ different river
strains of Atlantic salmon under a range of culture environments along the Norwegian
coast (Gjedrem et al. 1991). This work showed that there was little genotype x
environment interaction, the best families grew well under all conditions, and that
most of the genetic variation resided within rather than between populations.
Therefore the breeding programme was setup using a synthetic strain that combined
the best families from a number of rivers. The history of the Norwegian salmon
breeding programme has been described by (Gjedrem et al. 1991; Gjoen and Bentsen
1997; Gjedrem 2005). Today these breeding programmes are run by private
companies Aquabreed and Salmobreed. These programmes have a number of
similarities in that they utilise family units to ongrow individual families of salmon
until the fish are large enough to be tagged (PIT) a proportion of these fish from each
family are retained as potential broodstock and the remainder are ongrown under
commercial condition and their individual and family performance are assessed for a
wide range of commercial traits.( Growth, sexual maturation, body conformation) In
recent years additional traits such as disease resistance (IPNV, Aeromonas, ISA) are
also assessed in these families under controlled challenge conditions and this
information is also included in a selection index. The estimated gains in the
Norwegian programme are between 8-10% per generation (Gjoen and Bentsen 1997).

Similar programmes have also been developed in other salmon producing countries
(e.g. see web sites for Landcatch Natural Selection in Scotland and Chile,
Stofnfiskcur in Iceland, Aquachile in Chile). Today most farmed Atlantic salmon eggs
come from scientifically managed breeding programmes that have significantly
improved the performance of this species.
Need for Genetic Management of Broodstock fish.
The vast majority of farmed fish worldwide are still not under managed programmes
but under a variety of broodstock replacement strategies. The domestication of aquatic
organisms usually follows a set pattern with either the initial collection of young fish
or mature adults from local populations of the wild species. Once the farmers have
managed to control the growth and reproduction of the species then seed supply from
hatchery produced fish is usually preferred as this reduces the risk of introducing
diseases from the wild and reduces variability in the fry as they are likely to be the
same age and size. The decision to close a hatchery population, stop using wild fish as
broodstock, needs to be managed as it will be critical for the long term viability of
that particular population. The early domestication process and husbandry methods
can result in a dramatic reduction in the actual number of wild broodstock that
contribute offspring to the replacement broodstock pool (Effective Breeding number
Ne). The reproductive success of the original wild fish and the survival of their
offspring can be highly variable and without some form of tagging it may be
impossible to identify the Ne of the hatchery strain. Ne is particularly difficult to
estimate in marine species that mass spawn. Recent studies using genetic markers in
mass spawning species such as seabream (Brown et al., 2006) and cod (Herlin et al.
2008) have shown that the offspring of a few individual fish can dominate in potential
broodstock replacements. Even in species that can be stripped but still have high
hatchery mortality, so fry batches are frequently pooled, the Ne of the hatchery
population can still be dramatically reduced in species such as halibut (Jackson et al.
2003).

Message
Selective improvement works in aquatic organisms and improvement has been
measured for a wide range of different traits in many commercially important
species.

The potential improvement in a trait will depend on the initial levels of additive
genetic variation present in the strain(s) and how well they are managed in the long-
term.

It is never too early to start the genetic management of a new farm species.
Identification of potential source material and comparative testing of strains can
result in major improvements in performance and ensure genetic variation is retained
for future improvement.

The use of tags, genetic markers or some form of line management is required, no
matter what type of selection approach is used, to minimise inbreeding and
associated loss of genetic variation.

Chromosome set manipulation.

The plastic phenotype displayed by many aquatic species and the lack of genetic
imprinting in many invertebrate and lower vertebrates enables a range of genetic and
environmental manipulations to be undertaken in these species that can have profound
effects on their subsequent performance. These manipulations are not seen as genetic
modification as many of the outcomes have been observed naturally in wild fish
populations.

External fertilisation in fish presents a unique opportunity for geneticists to

manipulate the gametes to generate structured breeding groups or novel genotypes in
ways not possible in other farmed animals. One of the most powerful technologies is
the ability to induce parthenogenesis and derive offspring from wholly maternal or
paternal origins (see Komen and Thorgaard (2007) and Dunham (2004) for reviews of
these technologies). These technologies are achieved by destroying the nuclear DNA
in either the egg or sperm, using ionising radiation (Gamma or UV); the treated
gamete is then fused with an untreated sperm or egg, respectively, to produce a
haploid embryo. The haploid embryo could continue to develop but would normally
die before hatching. Haploid embryos make a good resource for gene-mapping as they
are effectively large single gametes that contain enough DNA for gene-mapping
purposes (Kocher et al. 1998). However, it is possible to make the embryo diploid by
inhibition of the second meiotic division or first mitotic division; such individuals
retain a duplicated set of chromosomes from the untreated gamete. Eggs fertilised by
UV treated milt can become meiotic gynogenetic offspring by shocks that interfere
with the second meiotic division, causing the retention of the second polar body. If the
shock is delayed to the first mitotic division then two haploid copies of the maternal
chromosomes are retained to produce a mitotic gynogenetic offspring. Meiotic
gynogenetic offspring are on average homozygous at 50% of their loci because they
retain a pair of chromosomes, sister chromatids, which have just undergone
recombination. Mitotic gynogenetic offspring are 100% homozygous because they
arise from the duplication of a single chromosome set and are described as double
haploid (DH) individuals. Eggs treated with UV or gamma radiation, to destroy their
nuclear DNA, and then fused with a normal sperm produce a haploid embryo that can
be made diploid by a disruption of the first mitotic division to produce an
androgenetic DH that is 100% homozygous.

Double haploid individuals are homozygous at all loci but different DH individuals in
the same family will be fixed for different alleles at any given locus depending on the
recombinant event that generated that gamete. Double haploid individuals from either
a gynogenetic or androgenetic background can be used to generate clonal or isogenic
lines as all gametes produced by an individual will be identical, even after
recombination. When these gametes are used in a second round of parthenogenesis
all of the offspring will be identical and clonal. It is therefore possible to generate
clonal lines in as little as two generations for any particular species or strain of fish.
Apart from the models species such as zebrafish (Streisinger et al 1981) clonal lines
have been produced in tilapia (Müller-Belecke and Hörstegen Schwark 1995: Hussain
et al. 1998 and Sarder et al. 1999) rainbow trout ( Scheerer et al. 1986 and Thorgaard
et al. 1990. Quillet et al. 2007) and common carp (Komen et al. 1991; Bongers et al.
1997)

If eggs are fertilised with normal sperm and similar shock treatment are applied then
an early shock at the 2nd meiotic division will generate an embryo that contains 3
chromosome sets and is triploid (3n). A late shock at the first mitotic division will
retain two diploid sets and produce an embryo with 4 sets of chromosomes and is
tetraploid (4n). These types of animals with extra sets of chromosomes are known as
polyploids. Polyploidy is lethal in higher vertebrates and birds but has been widely
applied in plants and aquatic organisms (Chourrout et al 1986).

The disruption of the meiotic and mitotic divisions can be achieved using a variety of
shocks, most commonly, hot and cold temperature shocks and pressure shocks
although anaesthetics and chemical treatments can also be used. Optimisation
experiments are needed to determine the timing of the onset of the shock, be it the 2nd
meiotic or 1st mitotic division, at a given ambient water temperature and the strength
and duration of the shock to maximize the effect for a given species. Dunham (2004)
has produced a list of over 30 different fish and shellfish species and the published
optimised shock treatments and outcomes.

Polyploid production in shellfish is different to that observed in fish because it is

possible that both the 1st and 2nd polar body are still present after fertilisation (Tian et
al. 1999 a,b). This means that a range of other polyploidy states may be present in a
given experiment to produce triploids (aneuploid, triploid, tetraploid and pentaploid)
in Pacific oysters depending on whether the treatment impacts on the 1st , 2nd or both
polar bodies (Stephens and Downing 1988; Guo et al. 1989). Essentially the shock or
the chemicals used interferes with the normal polymerisation of the microtubules that
control the organisation and separation of the chromosomes within the nucleus during
meiosis and mitosis.

The shock treatments used to produce 3n fish can have a long-term impact on the
viability of the fish as many of these treatments are at the thermal limits of the species
involved and often when carried out on a large scale under farm conditions the
treatments can be sub-optimal. One possibility is to use gametes from 4n fish.
Tetraploid fish having a balance number of chromosomes can still undergo meiosis
and produce viable gametes, which are potentially already diploid. So a cross between
a normal egg (1n) and a milt from a 4n male (2n) will generate 3n offspring directly.
This has been successful in rainbow trout (Myers et al. 1986) once eggs capable of
being fertilised by the larger diploid milt have been identified.

These techniques have been applied on a wide range of aquatic organisms (see
Dunham 2004). Direct applications in aquaculture include the generation of triploid
fish, usually from all-female strains, in a number of species in which sexual maturity
can reduce the size and quality of the farmed fish. Triploid females tend to be
effectively sterile as the unequal chromosome number interferes with the meiotic
divisions needed to produce viable eggs resulting inthe ovary of such fish being small
and containing few if any developing ova. This technology has been applied to the
production of larger rainbow trout (Bye and Lincoln 1986), channel catfish (Wolters
et al. 1982), common carp (Basavaraju et al. 2002) and in other aquaculture species in
which the onset of maturity in diploid strains slows growth. In shellfish the triploidy
is now widely used to improve the growth rate and improve flesh quality in oysters
(Allen and Downing 1988; Guo et al 2000). It can also be used as a biological control
mechanism to stop spawning in exotic species, such as grass carp used for the control
of aquatic weeds (Shelton and Jensen 1979: Wattendorf, 1986). To reduce the risk of
introgression of genes from stocked or escaped farmed strains into native populations
of the same species (e.g. Atlantic salmon in N.Europe and Eastern USA and Canada
e.g. Brown trout in UK, rainbow trout and cutthroat trout in USA and Canada)
(Kozfkay et al. 2006). Triploid sterility has also been put forward as a means to stop
the introgression of genes from transgenic fish strains that might escape into the wild
(see review Mair et al. 2007).

Direct applications of gynogenetic or androgenetic techniques on production fish is

rare and these techniques are usually undertaken in research laboratories. However,
these techniques can be used to generate unique genotypes that can be used to
generate new strains. The development of YY male tilapia used to produce all–male
XY tilapia can be generated directly by androgenesis as any male offspring in O.
niloticus will have the YY genotype (Myers et al 1986). Alternatively, eggs produced
by neo-female (males sex-reversed to female phenotype) and crossed to normal
males XY x XY will also generate XX females and XY and YY male offspring (Mair
et al 1995). However, all the male fish in such crosses have to be progeny tested to
identify their genotype. This approach has also been used in the development of all-
female fish (Pongthana et al. 1995). In the silver barb (Puntius goniontus) females are
the preferred sex as the ovaries are a delicacy. Meiotic gynogenetic offspring can be
produced in large numbers and are all-female so can be sex-reversed to generate neo-
males without the need for progeny testing. These neomales fish are supplied in large
number to the hatcheries as broodstock for the commercial production of all female
fry (Pongthana et al. 1999).

Gene Transfer Technologies.

The large number of gametes and the ease of collection, the control over the timing of
fertilisation, the ease of manipulation because of the large size of the ova and in vitro
incubation encouraged the application of gene transfer techniques in a number of fish
species. Particularly so when you compare the relative ease of working with fish eggs
to the complexity involved in mammals (Powers et al 1992). It was the much
publicised work by Palmiter et al. (1982) and Palmiter and Brinster (1986) that
showed huge growth improvements in mice, induced by the integration of growth
hormone (GH) constructs, that stimulated much of the early work on fish. Transgenic
fish containing the human GH gene (hGHg) were quickly produced in several
commercial species including goldfish (Zhu et al. 1985), rainbow trout (Chourrout et
al. 1986; Maclean et al. 1992; Penman et al. 1990; Guyomard et al. 1989a.b), channel
catfish (Dunham et al. 1987) and Nile tilapia (Brem et al. 1988). These early studies
utilised the genes that happened to be available and spliced together constructs from a
wide range of genes and promoter sequences from different sources and injected these
into the cytoplasm near the nucleus in the fish egg .

The results from these studies suggested that less than 5% of the injected fish ended
up with a copy of the construct integrated into the host genome. The problems
included cytoplasmic amplification and expression of the construct that often resulted
in an overestimate of the success of the procedure. Integration of the constructs
appears to occur after the first cell division so that only a proportion of the cells
within the host will contain the construct resulting in mosaicism. Breeding from these
mosaic fish means that only those that have the construct stably integrated into germ
tissue will pass the construct onto their offspring. Even with the advantages offered by
fish the process of microinjection is time consuming and tedious and was resulting in
relatively low levels of success. Other techniques have been tried that would enable
mass transfer of constructs into the large numbers of egg available from aquatic
species. These included electroporation, retroviral integration, liposomal reverse
phase evaporation, sperm mediated transfer and microprojectiles (see Chen and
Powers 1990 for review). Powers et al. (1992) believe that electroporation offers the
best possibility for mass production of first generation transgenic fish.

The earlier work concentrated on the enhancement of growth through the use of GH
constructs. With the improvements in the technique and the move to better constructs
growth enhancement was observed in a number of commercial species including
rainbow trout (Guyomard et al. 1989; Penman et al. 1991) common carp (Chen et al.
1989; Zhang et al. 1990) channel catfish (Dunham et al. 1992) tilapia (Martinez et al.
1996; Rahman et al.1998, Rahman and Maclean 1999). These studies resulted in a
wide range of gains in growth performance, between 10% -500%. However, the most
dramatic improvements were observed in the work undertaken in Devlin’s laboratory
in Vancouver. This laboratory developed total piscine gene constructs that utilised
either an ocean pout antifreeze promoter (ocAFP) used to control a chinook salmon
GH cDNA or a sock-eye salmon metallothionein (MT) promoter controlling a full
length sock-eye GH gene. When successfully integrated these constructs resulted in
significant elevation of circulating GH levels (40X), particularly in younger fish
(Devlin et al. 1994; Devlin et al. 1997). Growth enhancements of transgenic
individual over controls ranged from 5-30 fold increase in growth up to 1 year of age
(Du et al. 1992; Devlin 1994, 1995) and successfully passed this performance onto
their offspring. The same construct had similar effects when used in other salmonid
species, coho salmon 10-30X improvement, rainbow trout 3.2-10X improvement
(Devlin et al. 1995a ; 1997b), cutthroat trout 10X improvement, a 6.2X improvement
in chinook salmon (Devlin et al. 1995a) and 3-6X improvement in Atlantic salmon
(Du et al. 1992; Devlin et al. 1997; Cook et al. 2000).

It is now clear that fish species respond very differently to the effects of GH,
salmonids appear to be very sensitive and this is probably related to their physiology.
Growth rate in salmonids is generally slow particularly when water temperature is low
and appears to be partially controlled by the levels of circulating GH. An increased
level of GH in transgenic salmon effectively uncouples this regulatory control
enabling the fish to grow rapidly in the colder winter months in comparison to
control. This early advantage is further magnified as these fish smolt earlier and the
associated growth enhancement of this process gives these fish a further advantage
that can be subsequently magnified (Dunham and Devlin 1998).

Warm water species such as tilapia and carp can grow rapidly throughout the year and
are probably not as reliant on GH regulation. Transgenic fish produced from selected
lines do not show the dramatic improvements seen when wild fish are used. Devlin et
al. (2001) produced transgenic individuals from a wild rainbow trout and a
domesticated rainbow trout strain. The wild transgenics showed a 17X improvement
over non transgenics, but they did not grow better than the fast growing non
transgenic domestic trout. Introducing the gene into the domesticated strain only
improved overall performance by 4.4%.

It must also be remembered that it can take 4-5 generations of breeding to develop a
stable transgenic fish line to obtain a single step improvement in growth performance.
It appears at this stage that selective breeding can, not only achieve the growth
enhancements but also improvement across a number of other commercially
important traits that will enhance the overall value of strains in a cumulative manner.
However, the large amount of functional genomic work being undertaken is already
identifying possible candidate transgenic genes especially in the area of improved
disease resistance (Dunham 2008). Increased resistance has been observed in channel
catfish and medaka transgenic for the lytic peptide cecropin B. Pleiotropic effects on
disease resistance have been observed in fish transgenic for non disease constructs
(Dunham 2008).

To date there is no authenticated release of transgenic fish for aquaculture production

because of the difficulties in assessing the environmental risk of any escapes. Any
assessment of the phenotype of a given transgenic strain under laboratory conditions
and extrapolating this to an ever changing aquatic environment is almost impossible
(Devlin et al. 2006). This is likely to result in transgenic organisms remaining in
contained facilities until we better understand their characteristics and we have
reliable means of biological containment Mair et al. (2007).

Molecular techniques

Genetic Markers
There has been continuous development of genetic markers since the 1960’s as our
ability to separate and visualise protein and more recently DNA molecules has
improved. There have been a number of reviews on the development of molecular
markers and there application in aquatic organisms (see Liu and Cordes (2004) also
Liu 2007). The development of marker technology has been critical to our better
understanding of the population structure and evolution of aquatic organisms and will
underpin the latest developments in aquaculture genetics and genomics.

There are essentially 2 classes of markers Type 1 or actual genes of known function
(coding sequence) and Type 2 or anonymous DNA segments. In terms of direct
application, to date, Type 2 markers and in particular microsatellite markers have had
a dramatic impact on our ability to manage farmed fish in recent years. Genetic
markers based on proteins (allozymes) have been widely used from the 1960’s and
has proved particularly useful in developing our understanding of the population
genetics of aquatic organisms and the subsequent management and conservation of
fisheries. However, the relatively low number of loci that could be studied and the
low levels of observed polymorphism meant that their use in mapping and parentage
assignment was very limited. The development of DNA based markers initially in
mitochondrial DNA and various other restriction fragment analysis techniques such as
RAPDs and AFLP require no prior knowledge of DNA sequence and have been
applied to identify high levels of polymorphism but these techniques are not easily
transferable across laboratories and the dominant inheritance pattern of the sequences
being studied reduces their utility (Liu. 2007).

It was the identification of a new class of microsatellite DNA (Tautz, 1989) made up
of small tandem repeat sequences (CA, CAT, GATA) e.g. (CA)10 would be a simple
microsatellite ( CA repeated 10 times). Identification of these microsatellite loci
initially required the development and sequencing of microsatellite enriched libraries
constructed from the DNA of the species being studied. However, today with the
widespread sequencing efforts being undertaken in many species many new
microsatellite loci can be identified directly from databases. In the better studied
species (such as salmonids, tilapia seabass, seabream and carp) large numbers of
microsatellite loci have already been generated for genetic mapping studies.

Why are these markers so useful?

The fact that they are analysed using a PCR approach means that we can use a very
small sample of tissue (scale, fin clip, drop of blood) to generate enough DNA of the
markers we are interested in without damaging the fish. We normally have a large
number of microsatellite loci available for a given species and would select loci that
are the most informative, those having a large number of different alleles, in the
population under study. Many microsatellite loci can have between 10-50 alleles in a
given population. Although there are a large number of different alleles in a
population any individual fish can only have two at any given locus. These can either
be the same making the individual homozygous or different making the individual
heterozygous at that particular locus, simple Mendelian genetics. Therefore, if you
have a set of parents that you can genotype accurately at a number of loci (say 10 loci
with 10 alleles each) all of these parents should have a unique subset of 20 alleles
from the 100 in the population. If you then breed from these parents it should be
possible to predict the genotypes of the offspring arising from any given pair.

Several studies have shown that it is possible to assign a parentage to an untagged fish
grown under commercial conditions with varying degrees of success (Ferguson and
Danzmann 1998; Jackson et al. 2003; Norris et al. 2000). The success is higher in
controlled breeding , closed system, where the exact parentage of each cross is known
and in the quality of the microsatellite loci and PCR reactions have been optimised to
reduce genotyping errors. Success is usually reduced in uncontrolled mass spawning,
open system, in which any individual male could potentially spawn with any
individual female giving rise to a large number of potential families and the use of too
few poorly optimised loci. Simulations based on the genotypes of the parents often
suggest that high levels of assignment can be achieved with relatively few loci (4-6)
but in reality errors associated with PCR, the fragment analysis technology and
mutations in the locus or PCR primer site requires more loci to be screened to achieve
high levels of assignment (Liu and Cordes, 2004; Duchesne and Bernatchez, 2007).

Genetic mapping

The large number of type II genetic markers available in many species has to a great
extent been driven by groups interested in the construction of linkage maps. (See
websites listed). To date most of the maps are low to medium density with an average
map distance of between 2-15 centiMorgan (cM) between markers (a cM is equivalent
to a recombination rate of 1% between adjacent markers and is equivalent
approximately to 1 million bases). The process of developing a linkage map is very
well described by Danzmann and Gharbi (2007) in this review they also give a list of
all mapping efforts to date in a range of farmed aquatic organisms. The availability of
a linkage map in a given species enables the potential identification of Quantitative
Trait Loci (QTL) or sections of the genome that contain a gene or genes influencing
important phenotypic traits. To date, a large number, over 14, QTLs have been
identified in the rainbow trout by the Danzmann group and collaborators (see Korol et
al. 2007 review). There is a steady increase in the rate of publication of QTL in other
species such as Atlantic salmon, tilapia and common carp. In principle the marker
needs to be within 1cM of the trait to have widespread application in populations
other than within the one used to discover the QTL. Most maps in commercial fish
species still do not have this level of coverage. A strategy that has proved useful in
Atlantic salmon is to take advantage of the large difference in recombination rate, and
therefore map size between the sexes, this is in the order of 1:5 in male : female
recombination rate (Hayes et al.2006). Therefore by using a limited number of
markers spread through each of the linkage groups discovered to date it is possible to
localise a QTL to a single chromosome arm, within approximately 20cM using male
parents. Once the QTL has been localised it is possible to undertake the fine mapping
using more of the existing markers or generating new markers within the linkage
groups of interest using the female parents. This strategy has been used successfully
to identify a QTL for resistance to Infectious Pancreatic Necrosis (IPN) in a
commercial Scottish strain of Atlantic salmon (Houston et al. 2008).

The above approach shows the great benefit, at this early stage in the development of
these technologies in fish for collaboration between a commercial breeding
programme using traditional selective breed approaches and molecular geneticists. In
that the breeding programme had identified a medium to high heritability for IPN
resistance in the strain and could identify high and low responding families with a
high degree of accuracy, enabling a highly focused approach to localise the QTL (Guy
et al. 2007). The majority of the QTL that have been published are for easily
measured performance traits but as the technology and tools develop combining a
QTL approach for traits difficult to measure on the breeding candidate such as flesh
quality (e.g.colour) and other metabolic traits (e.g lipid metabolism and adiposity).
The combined approach should result in improved overall selection response.

It is important to continue the development of ever more dense linkage maps (see
Danzmann and Gharbi, 2007) to enable the move from using a marker close to the
gene to identifying the gene or genes and the allelic variation that gives rise to the
enhanced performance. This is known as Marker Assisted Selection (MAS) and has
potential to help in the improvement of traits with low heritability, identify possible
candidates early, before maturity, and in traits only observed in one of the sexes
(Lande and Thompson 1990). Despite the potential, the number of examples of its
successful application in livestock is still low and the attitude of the established
livestock breeders to MAS was described as one of “cautious optimism” by Dekkers
(2004) in his review. The application of MAS technology will increase in aquaculture.
De Santis and Jerry (2007) have listed a number of potential candidate genes based
on what we know from livestock. They have concentrated on genes associated with
growth such as GH, GHR, ILGF, GNRH, leptin, myogenin and showed evidence that
these might play a role in fish. It is clear that this list can only increase as function
genomic studies become more common.

Type 1 Markers
It is the rapid development in the identification of Expressed Sequence Tags (EST)
that is now paving the way for the inclusion of Type 1 markers or actual genes being
mapped to a species. The inclusion of Type 1 markers in a map means that we can
compare the relative position of these genes between the genomes of different species.
In aquaculture this is particularly important as it enables us to compare our map poor
species with the map rich model fish or mammalian maps. Sarrpoulou et al. (2008)
have shown the benefit of this comparative approach by using recently sequenced
model fish species, fugu, medaka and stickleback, and comparing them with three
commercial species gilthead seabream, seabass and tilapia. This approach showed that
the stickleback was the best intermediary, with 58% synteny with the gilthead
seabream, and to use this information to compare seabream with the other two
species.

Expressed Sequence Tags are generated by single pass 3′sequencing of

complementary DNA (cDNA) derived from messenger RNA (mRNA) from tissue
libraries. The mRNA is present because a particular protein is being expressed in that
tissue. It can be seen that by extracting mRNA from a wide range of different tissues
and from different tissues in animals that are being exposed to a range of novel
environmental conditions or at different developmental stages it will be possible to
see which genes are expressed and how a given gene or subset of genes is affected.
Most large fish genomics project have included a component to generate large
numbers of ESTs ( see the web sites listed, many list the libraries used and the EST
generated in the respective species). In any given tissue many 1000s of genes could be
expressed and the common housekeeping genes possibly in quite large amounts. In
order to identify the gene being expressed it is necessary to sequence a strand of DNA
that is complementary to the mRNA and compare this to other sequences already
collected and identified and held by the various databases worldwide.

Sequencing of cDNA libraries can be random but this approach can result in multiple
sequences from common transcripts. Improvements in the development of normalised
libraries, multiple copy cDNA is selectively removed, so sequencing is more efficient.
Subtractive Substitutional Hybridisation (SSH) is another technique used to remove
cDNA common to a control sample and that from a challenged sample, so only
differentially expressed genes will be sequenced. The sequence is then BLAST
searched against all existing genetic data bases, hopefully identifying homology of the
sequence to a known gene in another species (see Liu, 2007 for detailed review of
these technologies).

Direct linkage mapping of ESTs is extremely difficult as we usually have no

information on the allelic variation present for a given EST in the population under
study. EST sequences can be checked for the presence of microsatellites, recent
mining of EST data base sequences has shown around 10% of ESTs contain a
microsatellite. It is necessary to have sequence from several individuals in order to
identify whether a given gene is polymorphic. EST sequence can be also be aligned to
identify Single Nucleotide Polymorphisms (SNP). These are point mutations that give
rise to alternative bases at a given nucleotide position within the locus. SNP discovery
is still very challenging in aquatic organisms as many EST libraries have not been
constructed to look for intraspecific variation at a locus within a population, which
will hinder progress in SNP discovery. SNPs are however very common within most
genomes but it has only been recently possible to visualise and analyse these single
base differences routinely with new technological developments. The background to
the various techniques of SNP discovery and genotyping technologies are described in
detail by Liu (2007). It is clear that the ability to identify and map large numbers of
SNPs and automation of subsequent genotyping efforts has lead to rapid development
and application of SNP technology in human and animal sciences. Sequencing efforts
in Atlantic salmon rainbow trout and Tilapia will mean that it should be possible to
identify large numbers of SNPs within these species over the next 5 years.
In species with better developed genetic tools EST can be labelled and used as probes
to identify clones in an arrayed (macroarray filter) large insert libraries (e.g. Bacterial
Artificial Chromosome libraries BAC) that contain the EST of interest. If the BAC
library has been end sequenced and the various BAC clones aligned the EST will be
part of a contig (several adjacent BAC clones) and its position will be known on the
Physical and Genetic map of the species (He et al.2007)

The genetic tools for aquatic organisms are not as well advanced at this stage and
BAC libraries are either not available or have yet to be fully sequenced and contiged.
BAC libraries are available in a few farmed species Atlantic salmon (Davidson 2007
also see cGRASP web site), tilapia (Katagari et al. 2001), seabass (Whitaker et al.
2006) and the Pacific Crassostrea gigas (Cunningham et al. 2006). The availability of
a BAC library opens up the possibility of positional or map-based cloning of genes or
QTLs. The identification of markers flanking a gene of interest or QTL means that the
appropriate BAC clones containing the markers can be identified and continged and
the clone containing the gene identified and sequenced a process call chromosome
walking. BAC libraries are also a useful as a physical mapping resource as individual
clones can be labelled with fluorescent dyes and then hybridised to chromosome
spreads of the species. A number of BAC based physical mapping projects are
underway in Atlantic salmon (Ng et al 2005), tilapia (Katagiri et al. 2005) and
Channel catfish (Xu et al. 2007). BAC libraries have also been used as a resource in
the sequence of several different animal and plant species. The BAC clones are first
physicall mapped to the genome and the overlapping clones that give the minimum
distance can then be individually sequenced and a full sequence constructed (see Liu
2007 for detailed review).

Direct mapping of ESTs is possible in species in which a Radiation Hybrid mapping

panel (RH panel) is available. Radiation hybrid lines have proved difficult to produce
in fish, one of the first successes was in the zebrafish (Geisler et al. 1999) and the
same group helped in the construction of the first panel in a commercial species, the
gilthead seabream Sparus auratus (Senger et al. 2006). RH panel mapping does not
require a polymorphism only knowledge of the sequence so the relevant primers can
be used for PCR to confirm the presence or absence of the given sequence in each of
the hybrid lines. Radiation Hybrid panels enable large numbers of markers to be
mapped quickly without the restrictions of the recombination based approaches.
Work is in progress to develop new RH panels for other species such as seabass
(AQUAFIRST). The development of more RH mapping panels in other aquaculture
species has been on hold as to some extent with the rapid development in the other
tools and the expectation of more genome sequencing efforts in aquaculture species
making the cost and time commitment in developing such panels unnecessary.
However, despite international species community efforts to get their species
sequenced few have been adopted making the likelihood of more RH initiatives likely
(Rexroad, 2007). The addition of Type 1 markers to maps allows comparative
genomic approaches to be adopted to help in the identification of possible candidate
loci. Candidate loci or QTL identified in model species and the loci associated with
them may identify regions of synteny in the less well studied commercial species.

Functional Genomics
The other driver for the discovery of ever more ESTs is the construction of microarray
chips that contain cDNA clones or more specific oligonucleotide sequences derived
from critical parts of the gene in question. These microarray chips can contain 000’s
of ESTs derived from the whole aninmal or more focussed subsets of genes specific to
a tissue or a biological function, such as response to disease. The work in salmonids
has progressed the furthest and a description of the development of the genetic
resources including the various microarrays and the protocols used in these types of
study are reviewed by Rise et al. (2007).

Microarrays can be used to study a wide range of potential traits of interest to the
aquaculture sector. To date there has been a great deal of interest in disease related
responses (e.g. Piscirickettsia salmonis (Rise et al. 2004), Aeromonas salmonicida
(Ewart et al. 2005), Ameobic gill disease (Morrison et al. 2006) immune response to
LPS challenge (MacKenzie et al. 2006), live bacterial vaccines (Martin et al. 2006)
and DNA vaccination (Purcell et al. 2006). Gyrodactylus species, Lindenstrom et al.
2003, 2006: Fast et al 2006)). Other traits of interest include growth responses in
transgenic salmon (Rise et al. 2006) and stress responses associated with handling
(Krasnov et al. 2005) and temperature (Vornanen et al. 2005).

With large numbers of genes being monitored for their expression under a range of
different conditions it is clear that this type of study may identify potential candidate
genes that have large effects on the phenotype and may be of commercial importance.
The QTL approach outlined earlier cannot presently give the levels of resolution
needed to identify potential candidate genes because of the low density of the markers
available in many species. Projects that integrate the QTL mapping approach
alongside global gene expression studies may well identify possible candidate genes
expression patterns that correlate with the traits being studied (Haley and de Koning
2006). Advance in our understanding of the disease resistance in aquatic organisms
will identify pathways that are responding to infection than can be enhanced such as
the immune system or supported so that the fish can recover much quicker from such
challenges. Other traits of major importance in aquaculture for many species include
the control and delay of sexual maturation. This can be to improve the reproductive
biology so that fish will spawn and produce high quality gametes when required
essential for a sustainable industry. The converse is also true in species in which
maturation needs to be delayed or avoided.

Web based resources

Many new genetic tools and technologies have had to be developed over the last 20
years driven initially by the needs of Human Genome Project to sequence and order
3000 million bases. This story has been told elsewhere (Ventner et al 2001). These
genetic tools and technologies have now been applied to sequence a wide range of
other important model species, including fish. The following are the webites available
for a range of model fish species.

Zebra danio (Danio rerio) (http://www.sanger.ac.uk/Projects/D_rerio/) :

Fugu, Takifugu rubripes) (http://www.fugu-sg.org):
Puffer fish Tetraodon nigrovirdis (http//www.genome .gov/11008305):
Medaka (Oryzias latipes) (http://dolphin.lab.nig.acjp/medaka/index/php):
Stickleback (Gasterosteus aculeatus) (http//www.genome.gov/12512292)

This review is essentially aimed at the application of genetics and genomics to

aquaculture and will be restricted to existing or potential farmed species. There have
been a number of projects that have worked with important aquaculture species in
recent years. These projects have in many cases stated that one of their main goals
was to generate knowledge that would benefit the aquaculture sector.

Atlantic salmon (Salmo salar)

Collaborative EU funded project called SALMAP that aimed to develop new markers
and better maps for salmonids (Atlantic salmon, rainbow trout and brown trout). EU
funded project called SALGENE that aimed to generate large numbers of EST for
functional genomic applications. Resources from these projects are now part of a
large international collaboration GRASP and more recently cGRASP that has
continued to develop libraries, mapping resolution and functional genomic resources
for salmonids. (http://www.cgrasp.org/). The Norwegian contribution to the
SALMAP and SALGENE projects and continued collaboration and national efforts in
salmon genomics can be viewed at (http://www.salmongenome.no/cgi-bin/sgp.cgi).
Recent projects based on these international initiative have utilised microarrays based
on the ESTs collected to study gene expression under a variety of disease challenge.
The GRASP chip has been used to study Immune and disease responses to
Gyrodactylus species, Lindenstrom et al. 2003, 2006: Piscirickettsia Rise et al. 2004:
Ichthyophthirius Sigh et al.2004: Amoebic gill disease Brindle et al 2006 a,b: sealice
and vaccination, Martin et al 2006, Purcell et al. 2006).

Salmon Breeding companies.

AquaChile (http://www.aquachile.com/spanish/inicio.html)

Aquagen (http://www.aquagen.no/eng/)

Landcatch (http://www.landcatch.co.uk/)

Salmobreed (http://www.salmobreed.no/SideMal01Eng.asp?ID=64)

Stofnfiskur (http://www.stofnfiskur.is/?modID=1&id=67)

Rainbow trout. (Oncorhynchus mykiss) The EU STRESSGENES project overall aim

was to identify in fish candidate genes associated with resistance to stress conditions
and thus provide the physiological and genetic basis for new marker-assisted
selection strategies. The rainbow trout has been selected as the focus of this project
for several reasons: it is an important aquaculture species throughout Europe; there is
a large body of physiological and genetic data available for this species; strains with
divergent responsiveness to stress are available; all the partners have worked
extensively on this species. (http://www.irisa.fr/stressgenes/) This web site is closed
to non members of the consortium.

The recent development of genomic tools, particularly microarray technology, allows

systematic gene expression analysis of biological material and provides an integrated
overview of the global response at the level of gene expression. Such information is
of major importance for identifying genes responsible for genetic variation in
response to stress and for further development of a sufficient number of molecular
markers, a critical requirement for marker-assisted selection schemes. The present
study proposes, for the first time, to apply this new functional genomic approach to
examine a complex physiological problem: analysis of the pattern of gene expression
at the tissue level in response to exposure to a stressor. It is proposed to examine gene
expression profiles in rainbow trout during exposure to a range of stressors typical of
those encountered in the aquaculture environment. This should lead to
characterisation of stress-responsive genes as potential candidate gene markers.

Tilapia The genomic resources available for Oreochromis niloticus and other cichlid
species can be viewed (http://hcgs.unh.edu/cichlid/#maps)

European Seabass. (Dicentrarchus labrax)The recent work undertaken by a

multinational collaboration (BASSMAP) to improve the genetic tools for this species
and can be viewed at http://www.bassmap.org/) This project developed and mapped
over ??? type II markers and developed ???EST and mapped ??? type I markers. It
also generated a 65K BAC library with 8X genome coverage. The second phase of
this project has been included in a new project that will continue to improve the tools
(RH panel end sequence BAC library) can be viewed at (http://aquafirst.vitamib.com/)

Seabream (Sparus auratus) Another EU funded multinational project

(BRIDGEMAP) to develop tools for the Gilthead seabream The resources generated
can be viewed at (http://www.bridgemap.tuc.gr ). This project developed the first RH
hybrid panel in a fish and used this to map ??? typeI and ???type II markers (Franch et
al 2006). The second phase of this project has been included in the EU AQUAFIRST
project. (http://aquafirst.vitamib.com/)

AQUAFIRST is an EU project that seeks to identify genes associated with stress and
disease resistance in fish and molluscs to provide a physiological and genetic basis for
marker-assisted selective breeding. It builds on earlier EU funded projects:
BRIDGEMAP, BASSMAP and STRESSGENES. To counter poor growth
performance, impaired reproduction and increased susceptibility to disease, growers
have increasingly used antibiotics and drugs. The development of selective breeding
programmes using genetic markers offers a means to reduce, if not eliminate, the use
of antibiotics.( http://aquafirst.vitamib.com/)

Halibut Hippoglossus hippoglossus and Senegal sole (Solea senegalensis)

(PLEUROGENE) (http://pleurogene.ca/index.php) This is a Canadian and Spanish
initiative to bdevelop genetic tools for these potentially important farmed marine
flatfish. Other flatfish sequence and genetic resources can be obtained via the
GENIPOL website.

Cod Gadus morhua Atlantic cod genomics and broodstock development in Canada
(http://codgene.ca/index.php)

Common Carp. (Cyprinus carpio) EUROCARP project

Genetic Libraries
NACEE (http://www.agrowebcee.net/subnetwork/nacee/)
NACA( http://www.enaca.org/modules/news/index.php?storytopic=18&start=60)
USA related activities
http://www.animalgenome.org/aquaculture/

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