polyploidy - case study 1 chromosome manipulation...

Part 2

Chromosome Manipulations

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Manipulations

Polyploidy - Case study 1

Chromosome manipulation technology

Gynogenesis and androgenesis – Case study 2

Cryopreservation of gametes

Oyster Culture

• In 1996 (FAO) over 1,200,000 tons of the Pacific Oyster (Crassostrea gigas) were produced in the world.

• Although it is a Japanese species it has been introduced in

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• Although it is a Japanese species it has been introduced in Australia, North America, France and New Zealand, always voluntarily;

• Why? Because it was the only way to enhance the production of oyster in these countries.

• Are there alternatives?

Oyster Paper

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YES!

• Polyploidization,

• Hybridization between closely related

MASTERS IN AQUACULTURE AND FISHERIESGenetics and Selection

• Hybridization between closely related species,

• Genetic selection

Attractiveness of Polyploidy

• Sterility:

– Reduced environmental impact of escapees;

– No diversion of energies towards maturation;

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• Faster growth

• Disease resistance

• Simple technique

Polyploidization in OystersWhat is it and how does it work?

• Various ways to do it:

– Suppress polar body I or II formation during

meiosis using cytochalasin B;

– Pressure shocks;

MASTERS IN AQUACULTURE AND FISHERIESGenetics and Selection

– Pressure shocks;

– Heat shocks;

– 6-DMAP treatment of eggs;

– Mate tetraploids with diploids.

Mitosis

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Meiosis

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Triploid Oysters

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Tetraploid Oysters

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Protocols for the induction of triploidy

• Allow fertilization to occur;

• Shortly after (e.g. 10 min in rainbow trout) treat eggs

in order to inhibit extrusion of 2nd polar body:

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in order to inhibit extrusion of 2nd polar body:

– Add cytochalasin B or heat shock or cold shock or pressure shock the eggs. Thermal shocks are easier to implement although pressure shocks have produced better and more robust results (more expensive equipment required).

MASTERS IN AQUACULTURE AND FISHERIESGenetics and Selection

•The standard method is to induce triploidy by treatment of newly fertilized eggs with CB to prevent extrusion of PB2 (Allen et al., 1989).

MASTERS IN AQUACULTURE AND FISHERIESGenetics and Selection

extrusion of PB2 (Allen et al., 1989).

•The alternative method, possible because of our development of tetraploid oysters, is by mating tetraploid and diploids (Guo et al., 1996).

Growth of Triploid vs. Diploid Oysters

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Problems with Triploids

• For many species triploids are not allowed by law

(e.g. sea bass in Europe);

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• Although sterile many triploids differentiate and

develop gonads to some extent (mosaics) , so growth

advantage is not always there;

PAPER

Growth Trials with Triploid Bass

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Triploid Hybrids

• Triploids where one diploid set of chromosomes comes from one species and one haploid set comes from another.– e.g. grass carp x common carp or rainbow trout x brook

trout;

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trout;

• Often show increased survival;

• Sterility is more sure;

• Can often reproduce the growth advantage of triploids without the mortality or deformity rates sometimes seen in triploids (e.g. coho x chinook salmon triploids)

Gynogenesis

• Diploid individuals with both set of chromosomes

from their mothers;

• In species with homogametic females it will produce

all-female lines;

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all-female lines;

• Two main applications:

– Sex control for the production of all-female lines in species where females mature larger than the commercial size;

– Rapid inbreeding for the generation of inbred lines (mainly useful for research purposes, but potentially also useful for the production of hybrids between inbred lines with resulting heterosis)

Protocols for Gynogenesis

• Sterilize sperm using radiation or chemical treatments;

• Allow fertilization to occur;

• Shortly after (e.g. 10 min in rainbow trout) treat eggs in order to inhibit extrusion of 2nd polar body:

MASTERS IN AQUACULTURE AND FISHERIESGenetics and Selection

– Add cytochalasin B or heat shock or cold shock or pressure shock

the eggs. Thermal shocks are easier to implement although

pressure shocks have produced better and more robust results

(more expensive equipment required).

• Treatment applied in the first division will produce partially homozygous diploids;

• A late treatment results in totally homozygous diploids.

MASTERS IN AQUACULTURE AND FISHERIESGenetics and Selection

Problems of Gynogenetic Lines

• Highly Inbred, with low survival rates, poor

growth, high deformities, etc.;

– This can be reversed if a gynogenetic, inbred

population is hormonally sex-reversed and then

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population is hormonally sex-reversed and then

mated with normal females. The offspring will be

all females and outbred.

• As a means of controlling reproduction there

is a risk that introduced males will lead to

establishment of an unwanted population;

Gynogenetic Seabass Lines

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Cryopreservation of Gametes

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The benefits of cryopreservation in aquaculture species include the

following:

1. Cryopreservation can be used to improve hatchery operations by providing sperm on demand and simplifying the timing of induced spawning.

2. Frozen sperm can enhance efficient use of facilities and create new

MASTERS IN AQUACULTURE AND FISHERIESGenetics and Selection

2. Frozen sperm can enhance efficient use of facilities and create new opportunities in the hatchery by eliminating the need to maintain live males.

3. Valuable genetic lineages, such as endangered species, research models or improved farmed strains, can be protected by storing frozen sperm. This could be critical for marine species such as shellfish, where valuable broodstocks must be stored in natural waters.

The benefits of cryopreservation inaquaculture species include the

following (cont.):

4. Sperm can be used in breeding programs to create new, improvedlines and shape the genetic resources available for aquacultureoperations. A dramatic example of this is in the dairy industry, which relies almost entirely upon cryo-preserved sperm to produce

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which relies almost entirely upon cryo-preserved sperm to produceimprovements in milk yields.

5. Cryopreserved sperm of aquatic species will likely become anentirely new industry within the coming decade. Large, highlyvaluable global markets for cryopreserved sperm of aquaticspecies are on the horizon.