FISH 7650 – Fish Genetic Enhancement – Fall 2009

Section 1
General Introduction:

x In any genetic enhancement project we need to set out the goals where we need to
identify the trait we are interested in developing whether it is for economic, ecological
reason and the percentage of improvement we seek to build up, while a plan basically
discusses the genetic tool we will use to reach our goal such as cross-breeding,
hybridization and others
x The general breeding formula: P = G + E + GE
o Where P is the phenotype or appearance we are seeing or getting
o G is the genotype
o E is the environmental effect
o GE is the interaction between the genotype and the environment
x This will allow us to say that although the farmer may be excellent at growing fish by
providing the best environmental conditions sometimes the fish is genetically limited and
in other cases the fish is very good while the environment is limiting Æ genetic alone is
not enough
x Most studies study weight gain, and dealing with weight is not very easy due to the
discrepancies in the readings that takes place during the sampling and measuring stages
some of which are:
o % WT gain – this has a bias to smaller fish since naturally a smaller fish can grow
or double its weight much faster than a big fish because it is relative
o Average daily weight gain – is how much it is gaining per day in grams or unit
mass, this on the opposite side favors the bigger fish since larger fish have a
bigger ability to eat and hence gain more weight faster
o Instantaneous growth rate – measures relative growth rate but also has the
problem in being bias to smaller fish because they are relative
o Multiple rearing – is a method where when we sample and see we have weight
differences we need to normalize or make the fish of equal weights and we do so
by putting the smaller fish at lower density and feed them more than bigger fish
and once we get them at the weight we need we proceed with the experiment at
hand
ƒ For this to work we should not have compensatory gain, where these
stunted animals that are put on a lower ration diet should not have the
ability to regrow very quickly after we give them back their normal diet
and make up the difference, mammals have the ability to do so, Fish have
not yet been seen to be able to grow quicker to catch up
ƒ NOTE: size grading in a genetic experiment should not be done, since we
will be doing selection and changing the allele frequency of the traits and
hence favoring the smaller fish since we removed the bigger ones of the
bigger trait
o Regression method – is a good method to help us correct for environmental
changes but none the less genetic factors still affect it since at initial size it is not
 only the environmental factors that are causing this size, it is basically a
correction factor that we use to correct for environmental factors, we can clearly
see that regression favors smaller or slower growing fish since it gives this effect
of slower growth to the environment and not to their genetic makeup and it pulls
down the better fish readings, making them more conservative:
ƒ Byx =

ƒ Y (corrected final WT) =

x A Strategy to use if we know we are going to have size differences we can set up an
individual experiment that will allow us to produce a new correction factor within ONE
GENETIC GROUP hence it will result in a factor who is solely showing the effect of
environment and not genetic since genetically they are all the same – it has been seen that
this regression factor is almost the same and stands at around 3.0 which means that for
every 1 gram difference in the beginning it will be 3 grams at the end of the experiment
x Skewness Æ a possible problem with multiple rearing, skewness is when we have a non-
normal distribution (a distribution within the population that doesn’t look like a normal
bell shaped curve) and hence we either have:
o A positive skewness with a curve that has a tail to the right, reflecting some
members to be of bigger size
o A negative skewness with a curve that has a tail to the left, reflecting some
members to be of a smaller size
x A skewness coefficient will allow us to see how skewed the population is, and it can be
positive or negative
o At 0 it is not skewed
o Between -0.7 and 0.7 also considered normal
o Between 1 and -1 it is slightly skewed
o Beyond those it is considered skewed
x Japanese experiments in 1950’s wanted to investigate this problem of skewness which
was common in carp, where after measuring the weight of the eggs and the fry it was
normal until feeding started and the skewness appeared
o If the fry were grown out in individual containers then no skewness was see
o When an artificial jumper (also called shoot, which is the individual which is
bigger) and artificial because it is put inside and not grown with it such as a
bigger gold fish, this outsider took the jumper nieche and the normal distribution
was seen
o Experiments later showed that feeding is involved since smaller food when used
in labs made skewness less and pelleted food made it more due to higher
competition over larger pieces of feed, same results of higher skewness where
seen at higher fish densities and lower feeding frequencies
 o Î hence the main effect of this was competition over feed due to the carps
smaller mouth Æ we can get a relation between the factor of feeding, frequency
or other and the skewness
o Experiments now done on fry put in hapas and not labs and fed pellets, meal and
no feed, where we saw opposite results of skewness in smaller feed because it use
to go through the hapas and hence larger fish only had the chance to eat it while
pelleted stayed longer and almost all the fish ate
x Still discussing the concept of skewness, we see that different species react differently to
feed treatments where for a certain specie:
x If fed – it will create a bimodal or 2 bell shaped distributions side by side, where
in this situation all the fish had the opportunity to eat the artificial feed that has
been given to them, but some fish were not trained to eat this feed and hence were
unable to utilize it although it was provided, hence one group ate the feed and the
other sustained and grew on the natural feed stuffs in the pond
x If not fed – we saw the normal positive skewed distribution due to the competition
of the natural feed found in the pond
Magnification effect:

x Is where we have jumpers, what is happening is that they have an initial advantage which
is magnified through competition regarding size, this is what is reflected in the regression
factor which states that the initial size difference will be multiplied by several factors at
the end which will give this magnification in size
x Major factors that affect magnification:
x Feeding method whether on % body weight or satiation (since it reflects the
degree of the competition that is happening)
x Initial size – the smaller the initial size the higher the coefficient factor
x Final size – the more the fish grown the bigger they will become hence the
magnification is seen more predominantly and hence the correlation is higher
x The initial relative difference will also reflect a higher regression coefficient
x Magnification is basically due to a few fish getting a small advantage early on which
magnifies later and this might be due to environmental effect but also genetic
x Experiment: on carp population with skewness:
x A carp population was grown, and then the jumpers and the lagers where taken
out and multiple reared to become the same weight and then these population
were divided into 2 experiments:
x A communal pond where both where put together and into individual ponds
where they were grown separately and the growth was monitored
x In the communal pond we saw that the jumpers had a 15% growth advantage over
the lagers while in the individual tanks we saw a 7% advantage of the jumpers on
the lagers when they were grown alone
x This will allow us to say that 7% of the growth difference we saw in the
communal ponds (which was 15%) was due to genetic since that was the only
difference in the individual cases and the rest (8%) was due to environment effect
which is basically competition that we saw in the communal tanks
 x Communal evaluation has an advantage which is the size space of the experiment is less,
we tend to also standardize the environmental factor since they are being grown together
hence we have no differences and since all fish are in the SAME environment we can
consider each fish an independent statistical member ‘n’ hence the ‘n’ value will increase
a lot and make statistics better than in individual tanks where ach one tank is considered a
replicate and not the single fish
x What we lack here is that is that we don’t know if this ranking will hold when
these fish are grown alone which is the type of data we get from individual testing
or evaluations
x Note that the catfish experiment in catfish done in auburn showed a correlation
factor of 0.88 to 1.0 between communal and individual, noting that the 0.88 value
was produced when feeding was done on a % basis which enhances completion
and the 1.0 is when we feed till satiation
Age Difference effect:

x The concept of age difference and genetic difference between different evaluated groups
can be discussed where in an experiment done on common carp regarding their color, we
had
o bb-blue / gg-gold / bg-normal / N- Normal (dominant)
o 3 ponds where created:
ƒ First Æ eggs laid on same day of bb X gg and we got bb / gg / bg
populations where the normal colored grew best, followed by the gold and
the blue where the worst and we should note that normal colored fish are
expected to have the best growth always because enhanced physical
characteristics usually tend to take away from the growth and survival of
the fish
ƒ Second Æ gg allowed to spawn one day earlier producing all gg / bb
allowed to spawn one day later and produced all bb, the results showed
that the gold has a higher growth rate than the blue and the survival rate
was 96% for the gold and 4% for the blue Æ showed that the one day
advantage had a huge effect on survival
ƒ Third Æ bb allowed to spawn one day earlier, so the genetically inferior
type was given an advantage of the day and the gg allowed to spawn one
day later, results showed that in the first 30 days the blue grew at a higher
rate than the gold but then the gold picked up in the later stage and
established its genetic superiority, noting that the survival was 96% for
blue and 4% for gold which was the exact opposite than the first
experiment Æ this will allow us to say that the duration of experiment
termination is essential to see results, and one day advantage gave inferior
fish better survival but not higher growth rate Î main assumption here
which is essential to explain why the survival numbers are so different is
that they assumed that we had equal number of fish at the beginning of the
experiment of each type and they had no way to verify this during the
spawning process.
o One more note on the experiment is that it was done in fertilized ponds which
enhances competition and hence the one day advantage in carp means that all the
 correct sized feed found naturally that they could eat (since their heads are small
and are restricted to very small pieces of food) where already gone and hence they
would have starved to death Æ hence this one day advantage is very important
Maternal effect:

x Is another environmental effect, which is the impact made by size and age of the female
and the conditions of her environment on the quality and gametes and the growth of
embryos Æ therefore in genetic experiments, past history of the brood stock is very
important, need to be of same age and same environment in which they were grown to
get good valid results Æ we should always seek to limit this maternal effect
x We can see these results or affect in several locations:
o Measuring RELATIVE fecundity (which is the amount of eggs per Kg of mother
fish) varied greatly where it is much higher for younger and smaller fish per Kg
again
o Egg size varied as well, where bigger fish gave larger eggs, larger fry and larger
swim up but these differences of the maternal effect went away after a while and
took 20 days in O.niloticus and 30-60 days in catfish and up to 150 days in trout,
these differences between species is due to their developmental or growth rates,
where although they same varied durations but they all come at the same life stage
for these fish
x This might have a genetic component, and if so we might be able to choose for it – and
hence when we discuss the genetic (G) factor of our phenotypic equation we can expand
it into several factors one of them is maternal heterosis : G = ? + ? + MH
Other environmental factors:

x So as we said we have many environmental factors and we have discussed weight in

depth, other factors include:
o Tolerance to low dissolved oxygen – the larger the fish the lower its tolerance
because as it grows its volume increases faster than the surface area of its gills
hence it becomes harder for it to get oxygen in
o Size related disease, where we have some diseases attacking smaller fish because
they are less immune or vice versa
o Body shape is another very important factor which is difficult to study in genetics
tests, knowing that the body shape changes during the growth in factors involving
head depth, width, body depth, caudal depth and others – some papers don’t
correct for these changes as the animal grows while it is easily correct for by
dividing it by the total length to get values as a percentage or we can use
regression for correction – actually to get the clearest data correction we need a 2
fold regression / small fish has truncate bodies and a high head depth to total
depth ration which decreases as the fish growth then increases again at sexual
maturity due to the sexual development, hence if we chooses a small fish thinking
that its small head is genetic it might simply be at a stage where it is so and then
change
 o Dressing % and fillet % - same as before where the larger the head, generally the
smaller the dress weight, but this dressing weight tends to increase then drop
when the sides starts to get bigger at sexual maturity
ƒ In experiments where we want to choose for traits for size, we tend to go
for the bigger fish where we might results in bias
o The effect of long lasting disease and stress on the fish, where when a fish is
exposed to low DO for long period of times this effect their vital systems and will
physically reflect in internal damage to the internal tissues and some will go
beyond recovery
ƒ When we are also choosing for disease resistance, we should start with
experiments where the fish has not been exposed to this disease so the
environmental component of immunity due to already developed
antibodies will not be there and form a bias and give it advantage
o Meristic counts (such as counting the number of traits, number of scales …) these
are usually greatly affected by temperature, where some people say that it is due
to genetic factors but we now know that they are highly effected by water quality
parameters especially temperature.
o Î all of this so far has went to explain how the environmental factors may affect
the phenotype and will hence help us understand the genetic factors behind it or
what it might be masking

Genetic Factors:

x We usually think if we are starting a new population that we seek to increase diversity
and we can do so by increasing the number of individuals as much as we can, but actually
we are better of increasing the amount of individual diversity within the same individual
and get a few individuals since variability is essential
x We usually care for genetic improvement OR we don’t want any genetic regression to
take place and loose what we have
x When we create a population with a 2 individuals the variation is bug, where the number
of gametes for 1 individual can be produces is related to the number of heterozygous loci
we have which is denoted by N and hence the number of different gametes = 2N where if
all loci are homozygous and hence N=0 then all the gametes produced are the same
x This genetic variation may or may not have anything beneficial to us, we usually tend to
pull away from using siblings because they have a lot in common (at least 50%) and
hence we are decreasing the possibility of variation which is possible
x Variations of phenotypes can be:
o Discrete – black or white, you are or are not (no in between or decimal values)
o Continuous – there are or can be subdivided such as weights or parts of
x Qualitative traits are discrete in most of the times and especially when they involve a
small number of genes which they usually do, (refer to graphs) these graphs show that
whether a trait is a in a dominant or non-dominance situation we do have discrete groups
of characteristics at a low number of loci involved but as we increase in number the
groups and distribution becomes more bell shaped
x Some definitions:
 o Dominant – allele has dominance where when that gene has observable effect
when present in only when chromosome, so only if 1 copy is present then it is
observed
o Recessive – to be expressed both loci must have it represented on both
chromosomes
x Different types of dominance traits:
o Complete dominance – where Aa or AA shows Black while aa is white and the
from Aa to AA we have NO units of change while from aa to A- we have a big
unit of change
o Incomplete dominance – we have 3 phenotypes: bb (white) Æ 1 large unit of
change Æ Bb (dark grey) Æ small unit of change Æ BB (Black)
o Over dominance – where the heterozygous genotype is the best and is the superior
one, where ss is disease prone / SS is sickle cell anemic and Ss is healthy
o Co-dominance – is where both alleles are expressed and not something in
between, it is where we can see both colors Black and white (striped for example
but not a grey mix of them) AA X BB Æ AB / some molecules that are inherited
like this include isozymes and micro-satellites
o Additive gene expression – is where we have 3 phenotypes but the change from
one to another are all large and of EQUAL value unlike the incomplete
dominance situation: rr (white) Æ Large unit of change Æ Rr (pink) Æ large unit
of change Æ RR (red)
ƒ Example of fish weight genetic potential (where numbers reflect the value
of the allele that infers that weight)
x 8g-8g X 10g-5g Æ 8g-10g (18 gram total since 10 + 8 additive) &
8g-5g (8 +5) For this Locus
x Refer to summary Diagram in copy book and draw here:

x Linkage – is when we have 2 loci which are on the same chromosome and are in close
proximity and act as a unit of change together and hence are said to be linked
x Epistatis – is the interaction between genes at different loci and not necessarily on the
same chromosome
x Hence a genotype or genetic component = Additive (A) + Dominance (D) + Maternal
Heterosis (MH) + Epistatis (I)
o P = A + D + MH + E + GE + I
x Hybrids can be monohybrids where they are heterozygote for 1 trait (Aa X Aa and get a
ratios of 1:2:1 for genotype and 3:1 phenotype) or dihybrid for 2 traits (AaBb X AaBb)
 with phenotypic ratio of 9:3:3:1 if we have no linkage in dominance case and we have
independent segregation

AB Ab aB ab
AB AABB AABb AaBB AaBb
(AB) (AB) (AB) (AB)
Ab AABb AAbb AaBb Aabb
(AB) (Ab) (AB) (Ab)
aB AaBB AaBb aaBB aaBb
(AB) (AB) (aB) (aB)
ab AaBb Aabb aaBb aabb
(AB) (Ab) (aB) (ab)

x If we observed a different set of values in 9:3:3:1 then this may suggest that we have
epistasis occurring, such as if we got a 9:7 ration it means that when either aa (of which
we have 3) or bb (of which we have 3) are in homozygous recessive form they are
preventing the other dominant allele from being seen. 3+3+1=7 and the dominant 9!
x The case of epistatis that we just saw of the 9:7 phenotypic ratio is called recessive
complementary epistatis and are called so because the alleles on the different loci are
helping each other express something and when we have recessive homozygous
genotypes covering them when we have dominant alleles
x This makes more send when we observe it in a chemical pathway scene where going
from a to B requires the gene at loci A to be expressed and going from B to C needs gene
at Loci B to be expressed if any of them is down then we will not reach the final stage
which will give the needed result, and we have no mid-case phenotypes where if we cross
one step it will be enough
x Another case, is where we have development of a phenotype by progression where
having a homozygous recessive trait in one stop will give a partial phenotype and not the
full type, such as B-A- is black where we have at least one dominant allele on each loci
while bbA- and B-aa has a homozygous recessive on one of the pathways at least hence
the chain stops
x Single dominant epistatis, where we get a ratio of phenotypes equivalent to 12:3:1 where
if we have a single dominant copy of the allele in the 1st locus (for example A) we will
get the required phenotype and the expression of the second locus (for example B) will
only take place when the loci at A is homozygous recessive
o A-B- & A-bb Î black
o aaB- Æ Red since B- codes red and it was allowed to be expressed due to aa
o aabb – white since bb codes white and aa being so allowed its expression
x duplicate dominant epistatis, is a very important pathway or system which usually
controls the basic and vital systems of the body because it provides alternative or backup
pathways, where if any of the 2 loci had one dominant gene at least present then the
normal phenotypes will be expressed and abnormal phenotypes will only be seen when
we have aabb where both are homozygous recessive, this will usually result in a
phenotypic ratio of 15:1
 o this can be seen as having 2 pathways that will allow us to get to the same result
hence if one was blocked the second can be used

Deformities:

x most deformities seen don’t have a genetic basis, and the majority are due to
environmental factors which specifically occur during the hatching stage, usually farmers
link them to genetics and specifically to inbreeding but that is not always the case
(although it is sometimes)
x an example is the Red tilapia or O. Mozambicus, which due to its very high fecundity it
increases a lot in density in a pond producing small fish which are commercially
acceptable in some parts of the world and not others, hence people tried to cross it with
O. niloticus to get the desired color at a high growth rate but it reflected in many
phenotypes which were hard to categorize and study Æ in an effort to simplify it the
phenotypes where divided into 4 distinct groups and the researcher was able to explain on
how these color traits where being managed
x another example is that of the scale patterns on the common carp fish where we have
scaled (SSnn) / Mirror (scattered scales SsNn) / Line (on lateral lines SSNn) / leather
(almost nude SsNn) and death was observed when we had the genotypes S-NN and ssNN
hence the N gene is called a semi-lethal allele because it causes death when in
homozygous and it is also pleitropic because it has adverse effects on several traits
although it is one gene
o this is a system of 2 loci epistatis, where the N gene controlled several traits from
survival, to scaling it also effects the oxygen tolerance and many other traits
o note that the best growing and surviving fish are those with no physical anomalies
and who are rather denoted normal which is the scaled type in this example
x a fish trait in tilapias which is genetically linked is the saddle back trait (indented back),
which is due to a bone which is missing that supports the back meat, experiments have
shown it is not a sex linked gene and it is the situation of a dominant case where
o SS (lethal ) / Ss saddle back and ss is normal and the S gene is called semi lethal
allele that may alter our phenotypic ratios due to the death they cause Æ this is
one of the easiest traits to correct for and it is usually so for all traits where the
desirable phenotype is controlled by a recessive gene that I sonly expressed in the
homozygous form where we simply take out those with saddle back and hence
have removed the genes for it
x Some definitions:
o Pleitropy – is when one gene controls more than one trait
o Penetrance – is when we have the genes that should produce a certain phenotype
but doesn’t always do so (such as diabetes)
o Expressivity – is when we have differential expression of these genes, hence they
will be expressed but at a different severity levels and different age
ƒ Saddleback for example has 100% penetrance but with varied expressivity
x Continuing the discussion on pleitropy of qualitative characteristics on tilapia aurea
where we are trying to transfer the red trait to tilapia aurea because they have a fast
growth rate and they have good cold tolerance, but after doing the back cross (which is a
technique we will discuss later) we observe that normal color have good viability as well
 as those in the heterozygous form but those with the red color have showed a reduced
viability by 60-70% and these included those colored pink black (PB) / Pink (P) and pink
red (PR), we observed also that the red gene is not only effecting the viability but also
growth rates where the normal colored ones had the highest viability and best growth
rates which is expected since normal is always best

Simple selection procedures:

x We will discuss here how to deal with single locus systems and how to control it, where
as previously observed with the saddleback syndrome that is a dominant gene that we
want to eliminate it was simple to do so because we simply removed those that expressed
the saddleback phenotype and hence removed all the alleles involved Æ we are basically
here selecting for the recessive genes and when we do so we say that the population has
been fixed
x Elimination is not easy when the trait is recessive because phenotype of recessive genes
are not seen in heterozygous forms and hence considered a carrier, can’t be seen, only we
can remove those of unwanted trait since they will be homozygous but carriers will still
be in the population, genetically testing them is very expensive and labor some and hence
when we cull those with the unwanted phenotype we are removing only those with the
homozygous genotype
o Here we seek to select for a dominant allele, if we want to do this we will see a
success of a certain value with every generation, where in these situations we set
out a goal of reducing or eliminating the effect of a certain trait after an X number
of generations
o (1-q)n = (1-q)o / { 1 + N(1-q)o}
o Where (1-q)n is the frequency of the trait or the single gene after N generations of
selection and (1-q)o is the initial gene frequency of the recessive allele that we
seek to remove
o REFER TO COPYBOOK example
o Note: these formulas used will have standard deviation because they are
mathematical averages and will be associated to standard deviations
o Note: if we have for example 4% albinos which are caused by a genotype of
recessive genes denoted aa then the frequency of aa = f(aa) = 0.04 and frequency
of the gene = f(a) = square root of f(aa) = ¥ DQGWKLVLVZKDWZHXVHLQ
the equation above
o Note: by selecting against a recessive allele it means that we simply remove the
albinos or those that we are sure they are homozygous for the recessive gene,
these will basically have the phenotype that we don’t desire (in this example the
albinism)
o Note, if we seek to find the number of generations to get a certain decrease we
follow this order:
 ƒ The percentages of decrease in a population are the genotype frequencies
that will be given, in this case of the unwanted phenotype
ƒ From these we will get the gene frequency by square rooting the given
percentage and then we use the formula:
ƒ N = 1/(1-q)n - 1/(1-q)o
ƒ For recessive genes this will usually be a high number of generations
required which reflects how hard it is to eliminate recessive genes from a
population due to them being carriers
x Sometimes it is very important if we want to get rid of a trait we can do something called
a progeny testing where we estimate the genetic makeup of an individual through
measuring the performance and appearance or other characteristics and based on these
value we decide what to do with the animal such as by characterizing him as superior and
this is usually what is done in the cattle industry in a case of recessive genes (noting that
recessive gene problems are currently not a problem in fish culture) – but meticulous
testing is essential for us to ensure that we have superior animal in all traits, we have 2
basic systems for progeny testing:
o Heterozygous Test – where we cross our unknown animal of the normal looking
form (which can be AA or Aa) with a known heterozygote Aa where we seek to
see if our animal is Aa so we can cull him, the Probability of NOT DETECTING
Aa is (3/4)n where n is the number of progeny that we get out of the cross since !
is the ration of normal looking animals we will get in a cross of Aa X Aa if our
animal was heterozygote as well. As the number of n (the progeny) increases the
higher the chance we will detect Aa since we have more progeny to possibly
detect aa since each progeny is an independent event and if we get ‘aa’ then we
will know that our animal is a heterozygote
o Homozygous test – is where we cross our unknown animal (which can be Aa or
AA) with a known homozygous (aa) which we would have and the probability of
again NOT DETECTING Aa is (1/2)n and this is so because if our animal is
heterozygote then half of the time the progeny will be Aa only (less than the
heterozygote !) and hence we will detect it faster
o Î when we compare these 2 methods, for a 6% chance of not detecting the
needed genotype (equivalent to 0.06) we find that to achieve this 6% of not
detecting we will require 4 homozygous tests or 10 heterozygous tests which is a
difference of 6 progenies
x Progeny testing is potentially very important in fish since they produce a lot of progeny
per cycle hence n is very large and we can quickly detect the unwanted genotypes and do
the needed eliminations
Strains:

x A strain is within a specie with a common history and origin and at least 1 trait that
separates them from other strains in this specie, within a specie the differences between
strains can be huge based on the relation or trait we are interested in
o Such as differences between tilapia from different sources in Africa differ in their
cold tolerance, growth rates and catch ability and many of them have a genetic
basis
x It is very important that we think before doing any practice in our grow out system
because we might be inducing a genetic pressure without knowing it Ex: when we leave a
few fish that have not been caught in a net for them to reproduce in a pond, we are
indirectly selecting for hard to catch fish because these are the ones who didn’t make it in
the seine
x Starting a new farm or starting to culture a new specie, it is best to use established
domestic strains which are best performers in this environment, and it is the 1st step in a
genetic improvement program and this will rapidly increase the chances of improvement
in the initial progress of the genetic improvement program, since it will match the closest
to our situation
x Note that when a wild strain is domesticated they will be exposed to many selective
pressures due to culture system such as type of feed, density, human contact where with
time an organism will be developed to suite culture system being used, this domestication
occurs even without intervention of an aqua culturist Æ domestication effect can be
observed as few as one or two generations after removal from the natural environment,
where this is clear and wide differences in performance in culture media between those
domesticated and just brought in
x Example a wild trout when put in culture will have a fright syndrome when its sees
humans and will dive low while a cultured trout will come up since it know it will be
getting feed
x In channel catfish we observed and increase growth of 3-6% per generation due to
domestication selection due to getting use to medium therefore generally domesticated
strains of fish usually have better performance than wild strains
x Conversely in natural environments the wild strains do much better because
domestication would have made the fish less fit to survive in the open nature and wild
fish will be less vulnerable to angling (catching by a fish and line)
o In such a situation a few weeks later the domesticated fish are eliminated from
natural environment due to competition
x Strain variation is also important since it affects many other genetic enhancement
approaches Æ the strain we start with will affect all genetic programs that we seek to
follow
In Breeding:

x Is the mating of individuals which are more closely related that the average individuals of
the population, it is basically the mating of relatives and mathematically can be called the
correlation between uniting gametes, where if gametes coming from non related
individuals then the correlation factor produced is 0 or VV
x This occurs when we have selective or non random mating and the probability for that
occurring is high when we have small breeding populations
x Note: usually inbreeding harms the performance and it is interesting to see how naturally
this is avoided where in salmon bed reproduction where we have a lot of fish, we would
assume reproduction is taking place in the form of an orgy, but it is actually taking place
in an individual level where after testing the individuals who mated and their offspring’s
we saw that mates chose each other and ended up being genetically (genotypic ally) very
different hence increased the chance for heterozygosis in the offspring’s which will
benefit them and this has been linked to pheromone production that certain genotypes
produce that attract complementary other genotypes and VV
x Inbreeding doesn’t change the gene frequency in a population what happens is that is
changes the number or percentage of homozygous individuals and this is done by
increasing it. Gene frequency doesn’t change because we have independent assortment
and the percentage of homozygous individuals increase and those heterozygous decrease
and hence the genotypic frequency varies
x It is not always necessary bad all the time where if good genes become homozygote in
form this will be beneficial for performance, this explains where some mild genetic
enhancement programs are a form of mild in breeding
x But inbreeding depression can occur and does occur most of the time where when
compared to crossbreeding which is basically the mating of unrelated individuals and has
basically the opposite effects of inbreeding, inbreeding tends to decrease growth rate,
survival, reproduction performance, vigor of growth and tends to increase deformities
and biochemical disorders which is opposite of cross breeding
x Best way to correct a problem of inbreeding is by cross breeding and usually results are
immediate
x The genetic basis for inbreeding is dominance linked to an increase in homozygosis and
decrease of heterozygosity linked to epistatis in the process which can be due to AA
(effect of 2 additive loci) AD (effect of an additive and a dominant loci) and DD (effect
of 2 dominant loci)
x Hence at this point we can expand the components that flow into genetic variation and
they are due to variation of additive genes, dominant genes, maternal heterosis and the
epistatic effects discussed:
o Variation = VA + VD + VMH + VAA + VDD + VAD
x The inbreeding coefficient which is denote by F tell you how much more homozygous an
individual is than the population average and it can vary from a value of 0 where they are
heterozygous for all traits to a value of 1 where they are all of homozygous traits / hence
F denotes the value of loci which have become homozygous due to inbreeding
x We need to a have a starting point for the measurement of its value (although we will not
be going through calculating it we need to know its components), if we don’t know the
history and lineage of an individual then we give him a value of 0
x Note: homozygosis is absolute where it can be either YES OR NO while the inbreeding
factor F can tell us an Average of how much an individual is more homozygous than
others and hence the value is relative
x To understand these relations we can illustrate them in 2 forms (refer to copybook):
o Pedigrees, reflect family trees where we see how a certain individual is brought
from a series of other individuals in the system
o Path diagrams, show how reproductions have occurred to have reached a certain
individuals and will allow us to calculate the relatedness of some individuals in
this family where we should state these basic instructions:
ƒ To see how 2 individuals are related we need to reach one to another
through arrows, each arrow we cross is denoted a ½½ value and they are
multiplied to each other and if we can reach the other individual in more
than one way then we calculate the value of each way and then sum them
all up
ƒ Using these methods we see that direct siblings are ½½ related while half
siblings are ¼¼ related , noting that these relatedness factors are averages
and direct siblings relatedness can range from 0 to 1 and half siblings from
1 to ½½
ƒ Note: identical twins can be due to an embryo splitting (most cases) but
can be a one in a billion chance of two sibling getting the same genes
during the production process
x Uses of inbreeding:
o Some people use inbreeding to develop strict inbred lines to simply use them
during cross breeds later on thinking that it will enhance the good characteristics,
but this doesn’t occur in most situations where usually the resultant vigor is either
lower than the both initial populations’ original characteristics or has simply
brought back the difference after the genetic inbreeding depression that took place
during the inbred line development
ƒ We should mention that this occurs beneficially every once in a while, but
tends to require many crosses to be made between inbred lines that usually
require hundreds of inbred lines to be first developed and then crossed
 o Usually some people also use it in a line breeding situation where we want to
amplify a trait we induce inbreeding to increase it amount in the generations to
come
o It can be used during gene mapping processes to make results easier to read
x Gynogenesis & Androgenesis: these are methods usually used to intentionally make
inbred lines where Gynogenesis is where we have all female inheritance and produces
individuals called Gynogens and Androgenesis is where we have all male inheritance and
produce individuals called Androgens, and this can be produced in 2 ways or methods:
o Method 1 Æ to produce a gynogene, we have the primary follicle of the female
with the first polar body and when conception occurs the sperm will go in and for
a split second we have a triploid embryo since we have 3 sets of chromosomes
coming from the 1st polar body, the 1st follicular and the sperm that just went in
ƒ What we can do is irradiate the sperms before they enter and we do so
using UV radiation and not gamma rays (the reason is that gamma ray
radiation will not destroy the DNA completely and may produce smaller
segments called super-numery chromosome fragments that may go in with
the sperm and in most cases still have the ability to produce male proteins
and hence the produced gynogen is not pure), after irradiation the DNA is
destroyed but the sperm can still swim and fertilize the ovum, where now
at this point we shock the ovum so that the 2nd polar body is not released
and hence we produce a diploid individuals with full maternal DNA but
this individual is not Homozygous because he has 2 chromosomes in there
and they are not a clone of each other - this process is equivalent to a
sister and a brother (2 siblings) mating
o Method 2 Æ is done by irradiating the sperm and allow the 2nd polar body to be
released and then block the first cell division of the embryo (the Cytokinisis
stage) and hence produce a diploid cell which is 100% homozygous but if the
same mother produces several of those, each will be 100% homozygous but not
identical to the others because each time the egg will have different genes hence
we have genetic variation
ƒ If we want to produce a population with 100% homozygosis and no
genetic variation, what we will do is do this 2nd method again for one of
the individuals of the above discussed case where now we will take an
already homozygous individual and do to it the process to produce a
gynogen while will be again 100% homozygote with no genetic variation
since the individual we used has only 1 form of the alleles since he is
already 100% homozygous
ƒ Note that this second method is very difficult since shocking at 1st call
division is usually lethal, in addition we have uncovered all deleterious
 (deadly) disease which only appear in the homozygous form and may be
lethal
x Androgen production is the same where we irradiate the egg before allowing the sperm to
enter and hence the sperm donated 100% of the nuclear DNA (noting that maternal
Mitochondrial DNA will still come from the mother and it is essential for life, this is why
this process is sometimes not successful since mitochondrial DNA may be destroyed
during the irradiation of the egg during the occurrence e of the process)
x Fish Sex Genes: fish can usually have either an XY system like man where males are
heterogametic XY and females are homogametic XX or they can have a WZ system
where males are homogametic ZZ and females are heterogamtic WZ
o In XY situation, when we do gynogenesis we will get all females while in
androgeneisis we will either get an X which is double to form XX which is a
female or we get a Y which is doubled to get YY which is a male and survives
normally
o In WZ system, when we do androgensis we will get all males but when we do
Gynogensis we can get Z and doubles to become ZZ and form a male or we can
get a W which doubles to make WW which is a female
x Clones produced from homozygous rearing with methods discussed above are genetically
identical but do have phenotypic variances due to the environment component where,
although the variation due to genetics and environment-genetic interaction is Zero but the
Ve is still present
x In some traits such as the growth we expect them to be very near in terms of growth rate
but rather than varying on the normal shaped bell curve we observe them to be much
more extended and this is because that due to environmental variations in the form of
micro-climates and since the animals are homozygous then they are not able to have the
genetic variation to compensate and overcome the environmental variations provided
hence the phenotypic variation become very wide
x In a cross between 2 homozygous clones (clones of their initial populations, where they
are not identical but where identical to the population they came from) we will produce
an identical population which is heterozygote form some traits, this resultant population
although genetically identical all to each other, there are variations but it is very narrow
due to this heterozygosis that is allowing it to produce a somewhat close phenotype
outputs since it is able to handle the different environments (refer to graphs in copybook)
x Therefore inbred populations may be more variable phenotypic ally due to magnified
environmental effect
x If we want to calculate the amount of inbreeding per generation:
o F / generation = 1/8(number of bred males) + 1/8(number of bred females)
o Hence we see that after each generation we will get a certain inbreeding
coefficient that we ADD to the previous generation to get the cumulative or so far
additions
 o The higher the number of males and females breeding the lower the inbreed rate
or %
x Therefore for each generation we plug in the number of males and females and we
calculate them and then we add them up
x When we have equal inputs from males and females we have the slowest rate of
inbreeding because if there is one individual mating more than others in a certain
population then the probability of his offspring mating later on will increase a lot
x Based on generated data where when inbreeding reaches 12.5% to 25% (equivalent to
0.125-0.25) then we have a good possibility that we might produce inbreeding depression
and a decrease in performance, this is a measure produced mathematically to show a
RELATIVE increase of homoztgosity than we had initially and it is not an absolute
measure of it, so in some cases an increase of 15% homozygoisty may not effect or not
reflect a depression since homozygosis was very low initially while in other cases it
might be devastating due to a higher initial level of homozygosity
o If 50 pairs mate each generation on a commercial farm we should not expect
inbreeding depression for 25-50 generations (usually commercial farms have
more than 50 pairs hence this is not a problem)
o If we are working with a population with a very high fecundity and hence one
female can produce millions of eggs then this will increase in breeding because
only a few fish are used for farm rather than a lot (regarding the breeding females)
o An example on channel catfish shows
ƒ 1 generation of siblings mating showed F = 0.25 –where one strains
showed 0% depression and another 16% depression, the one that didn’t
decrease should have had a much higher level of heterozygosis to start
with hence he had more to lose before depressing
ƒ 2 generation of siblings mating showed F = 0.375 – where one strain
showed 19% depression and the other 30% and hence both depressed
because we reached the critical F value of 0.25 or 25%
x In the more homozygous strain we saw a decrease in egg hatching, decrease in survival
and an increase in deformities, generally we saw families with depressed growth showed
depressed survival and depressed reproductive success
x An example in rainbow trout, also shows as the inbreeding coefficient F increases, and
nears 1 the percentage of depression increases, where linked to that they followed the
depression of each rainbow trout family that they produced and followed by seeing the
different characteristics such as hatchability, weight, survival where some characteristics
depressed more than other
x In these experiments we see depression of growth rates and other properties die to not
maintaining good genetic principles but it is probably even worse since in these
experiments the depressions are under-estimated because some lines are so bad that the
whole fish died and hence we didn’t have any growth data for them and hence by
 eliminating the worst performers we have artificially increased the mean Æ also
experiment design of being in communal or individual ponds is important since if we had
them in communal ponds and the worst fish died then the other inbred species are now at
lower stocking density giving them a growth advantage
x In these experiments we must have a genetic control to be able to compare our results to
where this control can be:
o Random bred control – with no pedigree follow up, hence we have inaccuracies
because random bred populations will shift from one generation to another
o Inbred line controls – usually what we do is have an internal control in each
family line that we are testing and hence compare the performance line to its own
control hence being much more accurate
x We can generate the hypothesis that as homozygosis decreases the depression is
decreasing linearly until we reach a base, what actually occurs is that as heterozygosity
drops the depression will not occur since we have buffering and we have enough
heterozygosity to help us deal with it and at some point the depression occurs which is
bad Æ this situation is said to be buffered or canalized Æ this can be explained in terms
of biochemical pathways where the more homozygosity we get the more pathways are
being blocked until depressions occurs (like epistatic explanation)
x An experiment done on this is by measuring heterozygosis of isozymes variability which
will act as a marker where we saw:
o When marker showed 21% homozygosity Æ bilateral asymmetry increased by
21%
o When marker showed 44% homozygosis Æ bilateral asymmetry increased by
69% Æ here we had reached accelerated depression which is the 2nd critical value
for homozygosis
x It is essential to increase breeding populations size and to follow pedigrees so we can
avoid or minimize inbreeding and we can do rotations of our breeding ponds by rotating
either the males or females from one pond to another hence preventing siblings from
mating – essential to have more than 1 brood ponds
x Remember Æ inbreeding problems always corrected by cross breeding which will reduce
depression to zero as well as the inbreeding coefficient to zero

Inbreeding VS Genetic Drift:

x Genetic drift has a completely different concept that inbreeding and in many times it is
confused with it and it usually results in the loss of alleles or genes in a population when
it is present in low frequencies and these occur when breeding population size is low and
hence they are lost due to random chance and this is due to the founders effect which is
also called random genetic drift
 x If we have a small population drift can occur for an allele of equal frequency to others
may be lost due to getting a certain disadvantage which is perpetuated and amplified later
on
x Effect population size = Ne = {4 X number of males X number of females}/(number of
males + number of female} noting that they only account those that are breeding, the Ne
is the number of fish actually breeding and accounts for the ratio of sexes mating, the
higher the number of one sex compared to another the more mating individuals we need
to get to the same value of effective population
x A table in the slides showed us what is the probability of a certain allele disappearing
from a population over time (over certain generations), where from this table we can get
the number of effective population required to maintain it, in the table the F is the allele
frequency of our rare gene and P is the probability of losing the trait and the value in the
table reflect the amount of effective population required to maintain it at that F & P value
Æ to get the number of males and females we can plug the Ne value in its equation

Genetic Enhancement Programs:

x we have both short and long term enhancement project, where it was a major
misconception that all genetic programs are long term and results are very far, but as we
apply a program then we will have incremental improvement on the way
x most short term programs include strain selection that we start of the farm with as well as
hybridization and cross breeding which may or may not get us improvement and are
considered short term
x at this point we need to make a standardization of terms where:
o Cross Breeding Æ will refer to crosses done intra-specific crossing or crosses
within a species
o Hybridization Æ will refer to crosses done inter-specific crossing or crosses
between species
x A nice thing with fish genetic is that they are more elastic meaning we can cross between
species and get viable and reproductive fish as well as we can get triploid which his not
lethal

Cross Breeding:

x Is the mating of unrelated individuals in contrast to inbreeding (Note: for exam get
differences between inbreeding & cross breeding g- potential question) and it can also be
defines as breeding between different breeds, strains and lines
x In both cross breeding and hybridization we seek to get positive or enhanced heterosis,
where heterosis is the average performance of hybrid or offspring’s when compared to
parents and it can be:
o Positive in vale – which will yield better results
 o 0 in value – which will give same results
o Negative in value – which will be worse
x The Classical equation of heterosis:
o Heterosis = (average of hybrids – average of parents)/(average of parents) X 100
o Ex: for a characteristics of body weight if parent AXA = 100 and BXB = 110 and
crosses are AXB = 150 and BXA = 75 then average of parents is 105 and that of
crosses is 112.5 and hence heterosis is 6% which is positive
o In this classical definition the mean of the hybrids represents the mean of the
reciprocal hybrids where we count that a cross between female A X male B is
equivalent to female B X male A and take their average and this is seen to be very
Wrong, since we have a weakness in the formula because AXB is not equivalent
to BXA (noting that in writing we write female X male so A X B means female A
crossed with male B) since although nuclear genomes are the same, we have 3
factors that influence the heterosis other than that:
ƒ The cytoplasm-genome interaction where the whole cytoplasm is maternal
ƒ The mitochondrial DNA which is all maternal
ƒ The methelation of the base sequences, where although the base sequences
are identical we have differential methylation in males and females
x Well for practical reasons if we seek to use these programs to enhance fish in farm and
commercial situations we should not give both crosses value since we don’t care about
the weaker performer since we are not going to use them in the farm, so we seek to get
the heterosis caused by the best parent with the best resultant cross and hence the new
equation of heterosis is produced:
o Heterosis’ = (average of best hybrids – average of best parents)/(average of best
parents) X 100
o This will show us specifically which are the best performers and will reflect a
higher heterosis
x Some generalizations – we usually see a decrease in heterosis effect with age, where if
we weigh an individual early on regarding Body weight trait, the effect will reduce with
age
x Some other different type of crosses we can do is:
o Between 2 breeds such as A X B
o We can also back cross where we take the F1 and back cross it to one of the
parents so we will be getting (AXB) X B where we do this in this situation to get
the higher maternal heterosis from the F1 since we might have found out that a
certain characteristics such as reproductive ability is linked to maternal heterosis
so we will enhance maternal vigor here
ƒ But we should note that in this example we will be reducing heterozygosis
and hence will decrease heterosis for non selected traits such as
reproductive trait which may be highly affected by maternal heterosis
 o To solve the problem above we can do a three way cross where we use the same
female of the first generation and cross it with a 3rd breed, hence we get the
maternal heterosis and reproductive characteristics required but enhance
heterozygosis and hence heterosis for others and this will be a 3 breed cross:
(AXB) X C
ƒ The problem with these crosses is that we can predict if they are going to
be beneficial or not
x Weather 2 strains will give positive heterosis and how much this enhanced performance
will be is called the combining ability (also sometimes called the nicking ability)
o (refer to chart in copybook)
o Where if we are developing an F1 generation we have different families that we
crossed using males and females combination such as A1 X B1 and A2 X B2 and
so on, if we found out that A3 X B3 gave the best results then these would have
the best combining ability, so (knowing that we marked these fish) we will take
female A3 and propagate it with a male from the A family to get a pure line of
female A3’s to be used and do the same with the male B3 and then these will be
our parent lines whose offspring are the very good performing A3 X B3 which we
will stock our farms with
o This is called recurrent selection where we reselect for individuals that have a
high combining ability if done to one of the breeds it is called recurrent
succession and if done to both breeds we call it reciprocal recurrent succession
(like our example above)
x We can also have 4 way crosses of (AXB)X(CXD) this may enhance productivity if it
was reduced due to previous cross breeding processes to gain some characteristics which
lead to a depression in production ability (like corn example) but it is hard to do since we
need to maintain 4 pure parent lines (A-B-C-D) and then 2 crossed lines
x The genetic basis of enhancement due to cross breeding has 2 common components to
inbreeding which are dominance and epistatis but also has an added genetic action which
is over-dominance (not found in inbreeding)
x 55 of strain crosses resulted in heterosis for body weight in catfish – note in addition
heterosis tend to decrease when we use wild strain in crosses such as WXW or WX
domesticated and the highest is with domesticated (DXD)
x In case of cross breeding to increase the growth rate reached a maximum of 30% over the
best parent in some species, with other species other than catfish we get similar results
where a value of 10-15% considered to be very well
x We also observe that heterosis is much more apparent at younger age such as heterosis of
fingerlings is seen at 17-31% while 1-9% at the age of older fish or food fish stage, this is
in most experiments or situations attributed to the natural growth rate or phases of fish,
where fingerlings will naturally grow fast then slow down where those with no heterosis
will try tot catch up but will not be able to
 x Combining or nicking ability varies greatly among strains in cross breeding, reciprocals
do not grow at the same rate regarding growth rate and other characteristic since AXB is
not equivalent to BXA
x The maternal effect is very obvious where using a certain strain of male or females made
a big difference, where we saw in most crosses that female channel catfish are almost
exclusively used in all hybrid inter-specific crosses since they infer good growth at early
stages
x We also observed in channel catfish a heterosis of success in spawning, where F1XF1
crossbred showed an increase of around 49% heterosis as compared to their pure lines
which were at 18%, but this heterosis decreased at an older stage with some spawning
rate seen later
x Often we will get heterosis for some traits using cross breeding which has relatively an
increased rate of success, cross breeding also has an increased probability of enhancing
resistance to disease
x Generally a good program, since 70% of crossed lines showed disease resistance
increase, other characteristics are improved such as fry survivability
x In a case of rainbow trout data showing that strains used are very important, where DXW
are less likely to have heterosis than DXD
x In species like common carp the % of heterosis is less after cross breeding, but that
doesn’t mean it is not effective, if we do enough evaluation for our crosses we will find a
better line to use for sure
x In oysters, cross breeding has not been very productive but now new development show
that heterosis may be occurring
x Nile tilapia cross breeding also hasn’t shown very successful results, but work on cold
tolerance has been done with increasing heterosis in 10-20% of the evaluated crosses

Mating Blocks:

x In intraspecific crosses, since we are dealing with the same species we assume that
reproduction is easy and in most cases it is, sometimes we observe mating blocks or
incompatibility between strains of the same species reflecting also low fry number, it was
seen to be a problem with the female strain used because it always had a preference to
males of their own strain due to behavioral incompatibility
x Just to note Æ If someone is devoted to a cross breeding program he should devote a
large space for maintaining brood stocks since we have many line to support and we need
to keep them separated

Interspecific Hybridization:

x Basically uses same genetic principles and we look for heterosis based on dominant and
over dominant epistatis occurrence
x So gar there has been seen many hybrids developed and evaluated for potential
commercial used, the 1st problem we have to overcome is that we have reproductive
isolating mechanisms which are naturally there to prevent species from mixing and they
are:
o Geographic barriers preventing different species from mixing
o Mechanical (not very common) but occurs in fish that have to copulate where we
have the key-and-lock mechanism which needs to be made possible between the
reproductive organs
o Behavioral differences
o Temporal differences, where some fish simply mate at different times and at
different temperatures so we have the challenge of making fish gravid at same
time (ex: striped and white bass)
o Cellular incompatibility, related to mechanical where sperm and ova are
incompatible
o Gamete incompatibility, where sperm can penetrate but embryo can’t develop
o Hybrid sterility, where 2 species can produce progeny but the progeny is sterile
o F2 breakdown, where species can mate and will produce hybrids that can mate
but after a few generations the reproductive ability drops or disappears
x Note that when we discuss improved traits we are still discussing individuals traits but it
is essential for us to identify the overall genotype across the board
o Another note, almost always F2 performs lower than F1 in both inter and intra
specific crosses, with F1 being much better and F2 having a mid parent value
x Some forms of blocks occur where AXB cross sis successful while a BXA cross isn’t or
VV, and this is what happened in sunfish regarding operculum colors, where in turbid
ponds they copulated because they didn’t see each other (refer to example in copybook)
x Another example is the cross between a Brown and brook trout, where brown have a
developmental duration of 42 days but brook of 30 days when:
o Brown (female) X Brook Æ the hybrid developed in 36-38 days (mid value) but
hatched at 42 days because mother controls hatching and the brown continue
cycle at 42 days hence we had viability
o Brook X Brown Æ the hybrid developed in 36-38 days but hatched in 30 days
because brook develop to 30 days and this is controlled by mother, hence hatching
occurred before development finished therefore death occurred
x In general inter-specific cross hybridization is a failure to genetic improvement with a
chance of heterosis being very low especially for growth rat e(exceptions do happen)
o For example in the north American catfish we have 7 species, with over 50
crosses evaluated, only 1 showed heterosis (level of 2%) which was the Channel
X blue, hence probability of occurrence is very low but if it does occur then it has
very good impact
x NOTE Î is harvest ability is important then inter-specific hybridization is the one of the
best programs that tends to increase angling ability and seining vulnerability.
x The supper hybrid channel females cross blue male, were we have known that this hybrid
is superior only until recently it has been produced and that is due to its reproductive
isolation mechanisms discussed before that resulted in a low reproductive rate between
these 2 species even with induced or hand stripping spawning. Sometimes choosing a
specific strain of each will help this process
x Strain of parent specific affects the performance of the hybrid where we will always get
heterosis but the absolute performance of hybrids is related to the strains, an example is
the difference in blue strain resulted in a 40% change in heterosis value
x There has been cases where a study showed a channel catfish grew as fast or faster than
the hybrid or in some situation the blue did better Æ but this shows us that it is essential
to evaluate this research properly, where the channel strain compared to it here was
another strain different from the parental line and it is known to be a very good strain and
the one used in the hybrid was an inferior strain hence the result is not valid / these
comparisons are valid only when the hybrid is compared to its parental type strain Æ we
will always get that hybrids show better heterosis than their parental strains
x The better the strains we use the better the genetic value of the work we are going to get
in terms of hybrids, cross breeds or others
x In some cases we can’t separate from social aspects of consumer demand which will
effect why a certain hybrid is chosen over the other, some people are not using these
hybrids because they don’t have the blue strain to cross the channels that they have been
using, some farmers don’t like change and some people are not convinced which on the
other hand we have people hiding the technology to control markets
x The maternal effect was already discussed on growth but we also observed a maternal
effect on seinability and learning ability of the fish, we observed that the ratio of the
caught fish was changing over time where the channel and the white catfish saw a
decrease in catchability (most probably the fish learned how to escape) in the same
experiment over several times while the blue where always caught easily at the same rate
at different times, some of the hybrids showed each type of result but we observed that:
o Blue X channel Æ un altered seinability
o Channel X blue Æ decrease seinability Æ hence we see that it is a maternal effect
that we are getting which is varying our results
x In these reciprocal hybrids we see something also called paternal pre-dominance which is
external appearance of the value of behavioral and marestic traits for one hybrid is
statistically different from its reciprocal and more similar to the male parent
o Channel X blue Æ looks like blue
o Blue X channel Æ looks like channel
x When this was tested for cross breeding or intra specific crosses it didn’t show very
strong phenomenon expression
x This paternal predominance affects many traits different that those than the maternal
effect such as body conformation, uniformity of growth rate, harvest ability and other
x Telling the difference between hybrids and parents can also be seen with the morphology
of the swim bladed which is affected by the paternal pre-dominance
x The cause of paternal pre-dominance is still unknown but thought to be something
dealing with differential methylation of the genes from on sex over the other
x In salmonids, they have crossed almost everything which resulted in no improvement on
any level regarding growth hence no hybrids are used in the industry of salmon farming,
but we should note that in the situation where we get viable hybrids although we might
not get increased growth we will almost always higher total survival as well as enhanced
disease resistance
x The further the genetic difference between species the harder it is to form the hybrids,
where different species show differential results or improvement upon this
x Carp industry developed many hybrids but they are not used because of low heterosis
rates
x Tilapia hybrids, some of them are mono-sex male which we are interested in because
males tend to grow faster and we will not get the reproduction in the grow out system or
ponds to be a problem
o Enhancement with inter hybrids have also seen as enhancement of growth rate
and seinbaility, we should make sure of how the evaluation is done because if we
compare the all male hybrids (that naturally grow faster) to the parents which are
all male and female the mean of parents will be lower since it has females there
x Regarding the sex determining mechanism in tilapia
o In tilapia we always have the male fish sex genes dominating, and we usually
have 2 types of systems (mentioned before)
ƒ Where male is hetero-gametic XY and female is XX
ƒ Where the female is hetero-gametic WZ and male is ZZ
o In tilapia crosses we do a female XX with a male ZZ and get 100% XZ and since
the male genes are dominant then they are 100% males statistically
o This has been further confirmed by getting a ratio of 1:3 of female to male when
we crosses WZ –cross- XY
x However sometimes we get only 95-98% males while the others are females and this is
due to autosomal contamination, because sex determination is polygenetic, where we
have more than one gene or locus effecting the sex, where we usually have 1 main gene
that determines it and several others less powerful loci that may overcome the
designation of the major gene or locus
o These other minor loci are thought to be on autosomal loci and not on the sex
chromosomes
x In case of sunfish hybrids, they have showed an increase in growth with a large final size
but it minimized reproduction and hybrids (the F2) broke down and got a mid-parent
value performance
o Although sunfish showed heterosis for growth but since we use them in recreation
we don’t greatly care for the growth rate
x Referring to the GXE (genetic by environment) interactions, where what they did here is
evaluated several genotypes under different two or more environments, the used sunfish
the Green type which is known for rapid growth initially but slows down at the end and
the red-ear strain which has a slow start and then speeds up
o They did GXR and RXG crosses, where at a young age both hybrids didn’t show
heterosis
o At a later size of the fish they both grew to large than parent size and hence
genotype expression was affected by size (this is an exception since we usually
expect heterosis to decrease with size or age of fish or with time)
x GENERAL TREND Î this is essential to note that we have general trends varying
among species and strains where it has been observed in green and red ear sunfish (where
green are faster initial growers) and in catfish where white then channel then blue catfish
(in decreasing initial growth rate) has also shown the same trend
o Very high initial growth rate Æ will reach sexual maturity faster Æ growth will
be diverted into sexual development Æ maximum size is smaller
o Vice versa happens to the slow growing fish
x Hybrids of inter-specific crosses that were viable seemed to have much higher
survivability than parents
x Note that exceptions are found where hybrids don’t improve catch ability such as the
mean mouth bass (a cross between large MB and small MB) which counter the results of
the fishing experiment that show 82-88% of all hybrids where caught in a few days from
starting to fishing them
x For a hybrid to be beneficial it is not necessary that we have caused some heterosis to
take place, but rather if we get a general phenotype or genotype enhancement on all
aspects we are doing much better and this is considered to be beneficial – for example if
we are dealing with 3 traits, and the hybrid didn’t have any heterosis for any of them
compared to the best parent but mid range for all this may be a good intermediate level
for all characteristics
x For example:
o Striped Bass: fast growing / difficult to catch / heat sensitive CROSSED with
White bass: heat tolerant / easy catch and slow grower we get a fish which is mid
range for all hence more heat tolerant than striped but less than white and same
trend for all hence the general phenotype of resultant variety is very good
o Another is that Thai Walking catfish (desirable yellow flesh and slow growing)
CROSSED with African walking catfish which grown fast and has a pink/red
 color, the hybrid turned out to have enhanced growth when compared to Thai but
Thai’s color which is marketable, note that these fish are harvested at Thai size
although they can grow larger since consumers will not think it is that large pink
African type
x So far we have discussed results of F1 hybrids

F2 Hybrids & Backcrosses:

x We have discussed an example of those in one of the Israeli scenarios of crossing Nile
Tilapia with Blue tilapia at F2 level to enhance reproductive ability and cold tolerance
x Now we will introduce inter-specific back-crossing where we do these crosses for reasons
to do genetic mapping on one hand to link genetic markers to our information OR to do
genetic enhancement by basically transferring genes from one species to another in a
natural way and hence in some way can be considered to be creating a new species – this
has seen to be successful in some cases such as Russian sturgeon
o We do have problems with developing these backcrosses and are mainly due to
reproduction isolation techniques that prevent them from being mixed
x A back cross is basically crossing F1 generation fish with a parent such as the Tilapia
example where
o T-aureaus (with beneficial cold tolerance) X Red Tilapia (with demanded color)
to get F1 generation where then the red resultants from the F1 are re-crossed with
T-aureaus to even enhance cold tolerance more
o The resultant or backcross offspring may not show heterosis to the traits that we
seek to convey but in this particular example we did see heteroisis where it came
back showing better cold tolerance than T-aureaus as well as higher survivability
and lower lethal temperature
x An example on many backcrosses is seen in slide-198 where there is a table that represent
the different crosses, where specifically in case of catfish back-crosses the 1st generation
of these procedures showed very poor performance compared to the F1 and where very
close and even lower than the worst parent line

Selection:

x In the case of selection we can select for qualitative traits but we will now focus on
quantities traits where we have a range on phenotypes controlled by many loci
x Heritability denoted by h2 is so because it corresponds to the path diagrams we have used
before where arrows are squared while going from one point to another
x Heritability can be of 2 types:
o Broad sense Æ proportion in phenotypic variation is related to total genetic
variation = Vg/Vp
o Narrow sense Æ proportion in phenotypic variation is due to additive genetic
variation, and this is what we will be using most commonly
x We usually will select for dominance related loci and then we will get into additive gens,
what have been done previously in simple selection (like albinism and these traits) is that
when we wanted to eliminate a trait or so we did dominance linked selection but now
additive genes are used for most quantitative traits
x Most papers on heritability will discuss the narrow sense it is based on broad siblings
analysis which will include many components other than additive genes such as
dominance , maternal heterosis and others and it will tell us how much total genetic
variation there is but not specific or which type it is
x Heritability can have a value from 0 (where we have no additive genetic variation
therefore selection will not work) to a value of 1. In most cases if we have more than 0.2-
0.3 that means that we have significant differences and selection might work
x If broad since heritability is 0 the narrow is also 0, if it is not 0 then we know we have
genetic variation but we don’t know due to what and hence we can’t estimate if selection
will work (where for example if variation was all due to dominance for example we will
not have a good selection program)
x H2 = Variation due to additive / Va + M + V dominance + V epistatis + V environment +
V ge
x Now we have 3 broad levels or values of heritability
o Less than 0.2 is low
o Less than 0.3 is moderate
o Higher than 0.4 is high – means 40% or more of phenotypic variation is due to
additive genes
x If the heritability is low then cross breeding might be a better method to take than
selection while if it is high then selection would most probably work (not meaning that
cross breeding is not effective)
x Different populations and different generations within a population have different alleles,
different allele frequencies hence heritability values measured in different research
stations will give different values but they are usually close to each other
x Heritability measurements can be done in many ways:
o We can do full siblings analysis and based on that we can analyze the variance
and this will give us the broad sense heritability
o We can do the half-sibling analysis where we mate the male to 2 or more males or
one male to 2 or more females and this will allow us to get the effect or input of
additive, dominance and epistatis inheritance. Noting that a cross-factor design is
best where we mate one male and one female to many of the other sex
o We can also get heritability by doing regression of parent on offspring’s and we
can also do realized heritability estimates which are derived from actual
heritability experiments by selection and we measure progress of an actual
selection response
x These heritability’s can be Made relative to the Damn (female), the sire (the male) or the
Damn and the sire Æ refer to equations
x Note that these methods do have some weaknesses such as:
o For heritability relative to damn – we are multiplying the variance of the damn by
4 and this has a maternal effect component, if it was a large component then it
will affect our results
o In the heritability relative to the sire, we multiply the sire variance by 2 and this
usually have a dominant component hence we will me inflating it if it was large
x Best compromise is getting the heritability of the mixed S & D
x Realize heritability can also be h2= R / S
o Where R is the response or genetic gain and S is the selection differential
x The estimate heritability is easier to do since we don’t need any information of the
parents and we just given an estimate, while realized heritability is a much more difficult
process where we need information on parents, cross them and then evaluate the crosses
– we observe that estimates usually give values higher than realized heritability in catfish
and trout body weight increase
x As mentioned prior h2 varies between generations and in case we adopt a long term
selection program we want that to happen since this will change allele frequencies and
will help us get the best heritability h2
x Selection differential is the mean of selected parents – mean of control parents
x If we have additive genetic variation, those that are larger means that they are genetically
better, were if after selection and crossing it didn’t work then it might have been due to
other reason such as environment
x Refer to graphs in copy books to get values of how we get selection differential and
response
x Note that with time and longer generations in our experiment we will get less
improvement in each generation because we reach a plateau
x H2 = (mean of select progeny – mean control progeny)/(mean select parent – mean
control parent) = response to selection / SD
x Note selection differential is different from intensity of selection denoted ‘i’ where
o I = Z / P where Z is the height of the line at the point of truncations and P is the
area under the curve from the line of truncation to the end of the graph
o I will show us how strongly we are selecting, where if we select the top 1% is
stronger than selecting the top 20% and this will have implications on our
program
x Purpose of doing these half and full siblings tests or analysis is to tell us if a genetic job is
possible, if we conduct selection we can predict what possible results may be
x Progress or genetic gain or response denoted P:
o P = i.(standard deviation of phenotypic variation). Heritability (this will give us
how much progress we will see from one generation to the next)
 o P = (selection differential). Heritability
x Note we don’t always select the top percentiles due to a trend that we see:
o The less intense the selection although the slower the enhancement we see in the
few generations but in the long run over many generations it will have a higher
total response than more intense selection programs
o A intense program will be faster initially but will meet the less intense form on
some point and then will be less than it, due to more inbreeding and a smaller
pool of genes and sometimes a more intense selection due to use having less
individuals we may accidently loose a good allele before having the chance to
select for it

Section 2
Genetic Markers:

x We have a variety of genetic markers including mitochondrial DNA, isozymes, RFLP,

RADH, micro satellites, SNPS, AFLP with older methods as well such as blood typing
which is currently not being used anymore
x In case of restriction fragment length polymorphism or RFLP, usually it is the most
commonly done to mitochondrial DNA since the sequences is much shorter while it is not
very practical for nuclear DNA because it is very large
x A genetic marker is a protein product or a DNA marker or segment, essentially a genetic
way to mark an individual like branding
x Uses include: genetic linkage mapping, taxonomy, population differentiation,
contribution to different populations in mixed populations, determine genetic similarity,
detecting inbreeding, identifying species and hybrids, developing QTL maps (gene
maps), allowing studying the expression, micro array analysis, and is essential for marker
assisted selection as well as studying stocking effects
x Isozyme analysis is still very useful in studying population genetics and will allow us to
develop a map which is considered to be low density, an isozyme is a multiple molecular
form of an enzyme, considered to be a type of marker with important physiological
purposes, where although they are very small in number (regarding variety) they are still
used – hence for a certain enzyme they have one locus where they are found but isozymes
have 2,3 or more loci and these are due to evolving of the species where most started with
enzymes that acted on one loci only but with time due to mutations and recombination to
help body development or for it to be more efficient we will have some temporal changes
in the isozyme allowing them to function at different locations such as LDH has 3 sites in
the muscles, liver and eye
x Usually markers are of 2 types:
o Type 1 – actual markers from actual genes
o Type 2 – markers which are part of non-coding sequences
x We also have other than enzymes due to different loci we also have allelic variations
within 1 locus hence producing variants of the enzyme called allozymes (which are
different enzymes due to variation at 1 locus as oppose to several loci producing the same
enzymes such as in isozymes), these are inherited in a dominant fashion hence we can
determine the exact genotype of the individual for hat trait
x On a gel of electrophoresis we can study up to 80 individuals which can be slices and
manipulated to generate a lot of data points hence considered to be a very powerful tool
x To do enzyme analysis we do electrophoresis which is studying the movement of protein
molecules in an electric field and here we have specifically movement of DNA molecules
where we can have several types of gels which the best to use are the starch gels
x The gel is porous with somewhat uniform matrices depending on our DNA sequences
that are placed in the well, they come in different shapes and sizes and charges (different
allelic forms reflect different proteins and enzymes) hence these proteins tend to split
based on charge, size and shape and when we do this to DNA it is only done based on
size and not the other factors
x Now we have a gel where electrically has been run for it we need to stain to see the
separated proteins, this is called histo-chemical staining which in case of isozymes is
done based on specific dyes that will deposit on that protein location and identify it, we
place the gel in the tray and place the chemical solution and a reaction occurs (basically
involves providing the substrate of this isozyme) and this will trigger a sequence of
reactions where one of the steps involves formation of a color band
o If we are studying acid dehydrogenase, we provide it with lactate that will
eventually turn blue, sometimes these coloration are done by a negative reaction
where the dye will color everything except what we need
x Another criticism of isozyme analysis is that we don’t see a lot of variation when
compared to micro-satellites but when we have a large population and many species at
this level when we use it we see many differences very clearly
x In isozyme gels, we measure the relative speed of the sequences and we use a control
individual of known genotype and place them in this sequences and then we measure
relative speed of migration and we don’t use a size ladder
x Not all mutations are reflected in banding hence we sometimes have hidden variability, in
addition we should note that certain isozyme function differently in different gels,
switching gels and hence varying the pH will produce different results since pH has an
effect on the charge migration but these are not very common
x In a study around the world that supposedly studied the same tilapia species showed that
we have varied results and hence most populations where contaminated and actually they
were not studying the same species at all
x Measure of heterozygote, mean heterozygosis usually provided when we do isozymes
with mean heterozygosis tends to vary greatly between species – produced when we add
up all loci and dived them by observations
 x Isozymes are inherited Co-dominantly and that is why they are powerful in population
hence we can get the genotypes needed

Mitochondrial DNA Analysis:

x Circular DNA molecules of maternal inheritance, but we have a phenomenon called

paternal leakage which is very rare where mitochondrial DNA in sperm is usually lost
before penetrating the egg but in some cases this paternal DNA does go in and hence
contributing to the mitochondrial genome
x Mitochondria have energy and oxygen metabolism genes and these can usually be used
for analysis noting that some isozymes even have loci in mitochondria, in one cell we
have several mitochondria hence although from total genome they are less than 1 % in
terms of information but since they are found in many copies then they are very
important and helpful
x Due to mutations and errors this mitochondrial molecules and it having size differences
and polymorphism as well as mutations of certain restriction sites can allow site gain and
loss
o EX: Xba restriction enzyme recognizes sequences TCTAGA and when acted on
by 3 alleles A-B-C at the same size of 16.2 Kb where different alleles have
different numbers of these recognition sites therefore they cut at different number
of strips, different locations and hence producing different sized bands which can
be determined by electrophoresis
x One of the powers of using mitochondrial DNA, if we are trying to use ancestry of
individuals or if we are doing communal analysis with reciprocal hybrids being hard to
tell apart and since nuclear DNA is virtually identical then they can be differentiated by
mitochondrial DNA because ach have different mothers
x Example given here is of the Red breast fish which was seen to be having excessively
large size where it reflected the same appearance of the red breast fish but after
mitochondrial DNA showed that it was a result of a hybridization effect due to blue gill
mother since it showed blue gill mitochondrial DNA, and since morphologically it
doesn’t look like F1 then we know hybridization has took place in the past generation
x Note that genetic markers are very easily used to differentiate between 2 different species
but becomes much harder but still possible to differentiate between different strains
x The problem with DNA technology and looking at populations is that it is so powerful
that if enough work is done then we can separate each individual from the other due to
high ability to sequence individuals and others, it is essential for us to use technology
properly in terms of using common sense in choosing proper techniques in needed
situations
x RAPDs – randomly amplified polymorphic DNA is one of the genetic markers that can
be used where the levels of RAPD variation within strains is less than between species,
 advantages is that it is inexpensive, requires no upfront preparation work and shows
variation where most people are getting away from RAPD because of 2 major things:
o They are inherited with dominance hence we can differentiate between
homozygous and heterozygous individuals which is a weakness
o We also have a problem with repeatability of the experiment were as we vary
hybridization stringency we will alter results as well as band location and
densities, hence this Data many times is not accepted for these reasons but if we
are conservative when using them in our interpretation then our results can be
very good
x AFLP – next step up from RAPDs which is also becoming a techniques not used a lot, it
is slightly more difficult to do than RAPDs as well as slightly more expensive to do but
still very easy relative to theirs with advantages over RAPDs that it is more robust and
not a problem with repeatability of the experiment and hence better results and it
produces high levels of polymorphism, its disadvantages like RAPDs is that they are
inherited dominantly and they are both (RAPD and AFLP) type 2 genetic markers which
are found in non coding sequences technically what we are doing is amplifying a
sequences in the non coding regions and some will say that it is not really a locus that we
are locating and hence it will either show up or not regardless of the alleles found on it
x Microsatellite markers – much more superiors and these are simple sequences repeats and
they can be of both type 2 markers or type 2 where they can be repeated embedded
sequences within the gene and they have a lot of advantages including that they are
evenly distributed in the genome hence important for linkage maps and it is highly
polymorphic (where even 2 very similar individuals will at least on average have 2 allelic
variants here) and they are small loci markers and they are most importantly inherited co
dominantly, as for this disadvantages:
o Disadvantages include is that it is a much more expensive technology and more
technically challenging and requires more skill and talks a lot more work which is
done before analysis to develop these micro-satellites and make sure that they are
working, the reason we have these micro-satellites is due to the he variation that
occurs due to mutations that are called seepage events which are what makes
them so polymorphic and helpful BUT these excessive mutations makes it hard to
trace back in generations because the genotype changes constantly
o Micro-satellites since inherited co dominantly then we can easily assign parentage
with high accuracy and we can differentiate between homozygous and
heterozygous individuals
x EST – express sequence tags, good for certain experiments where partial cDNA
sequences and useful for looking at differential gene expression and they are type 1 gene
markers because they are produced by reverse transcriptase using a library that we have –
it will allows us to see what is the importance of a certain gene in a specific challenge
that the fish was put in because it will reflect higher copies of it
 x SNPs – single nucleotide polymorphism, this is the most accurate where it is basically
sequencing the whole genome section we are interested in, where every time we have a
sequence change in the form of a nucleotide it will be picked up as variation, these
changes may be beneficial or not
o They are defiantly inherited co dominantly because we seek both copies of the
nucleotide at each location, BUT it is very expensive and time consuming but
technology may develop to make it easier later on

Molecular aspects of genetics:

x A few examples on home molecular genetics had an impact on aquaculture, a lot of times
the role of molecular knowledge in aquaculture is rarely used because more of it is in the
form of breeding but example of it used as marker assisted selection where we have the
ability to detect markers that will allow us to fin genetic differences / transgenic which
involves the insertion of a fish gene and see how it will effect performance /
environmental impact of aquaculture on native environment and can be done by studying
genetic diversity and the loss of diversity in the natural environment due to the
introduction of certain species and we can also use this approach for disease studied that
are also important in identifying indicators for the occurrence of these
x We have the genome as a messy compilation of information only a small part of it is used
or at least we have interest in and the study of genomics will help us pull out the genomes
in discrete units that we can manage and make use of, so of all the DNA only some of it
is coding which we are interested in and then within the gene we have axons and introns
which are transcribed to an mRNA to see which genes are expressed under certain
circumstances and then the protein produced to do the actual job
x The central concept is changing DNA found in all cells into RNA which is the active
transcribed DNA which is what mainly differentiate one type of cell from the other
because certain cells are made into what they are by the types of genes which are
expressed in them and that will eventually lead to the production of specific DNA
x The observable phenotype differences can be due to genetic code differences due to
mutations which are beneficial or due to environmental selection allowing for
development of new strain and eventually species which are isolated reproductively to
ensure their continuity
o Using genomics seeks to estimate or to know what a certain genotype will be
reflected in interns of phenotype and this is what we are studying at the molecular
level
o We can also use some associated markers to these sequences that will allow us to
reach the traits that we want

PCR:
x Also called polymerase chain reaction is the technique allowing the amplification of
small quantities of DNA using thermal cycling and enzymatic reactions, it was developed
in 1984 (refer to the websites)
x In each cycle we double the copies using specific reagents which are the primers that are
specific to certain DNA fragments of our interest / the TAC polymerase which elongate
the sequences / the DNTPs which are the nucleotides that we add that form the basic unit
which is used to make the sequences larger and the buffer
x The process involves: putting the DNA sequences , then add primer one which is the
primer designed to attach of the specific sequences of interest then we add primer two
then we add the DNTP’s then the DNA polymerase (TAQ) is added which reads the
sequences and builds the new chain – note that the polymerase being used here is one
which is heat resistant to face the thermal cycles
x The thermal cycles tend to go through 3 temperatures:
o 95 C Æ the double helix is separated
o 50 C Æ the primers attach to the start sequences naturally
o 72 C Æ is the extension where the polymerase attach and start adding the DNA
nucleotides
o ONCE done the primers fall off and the cycle goes back up to 95 C
x Note that starting from the third cycle our main product starts to develop which is the
desired sequences which is limited from each sides by the primer that we are using
x Note that the efficiency of the PCR is directly related to how fast we can cycle the
temperatures
x We have 2 basic types of PCR:
o BASIC PCR Æ which is what we have discussed where we amplify short
sequences less than 10 KB sections and will help us isolate genomic DNA, or
disease diagnosis or fingerprinting as well as cloning it needs mainly the DNA
template, the DNTPs, TAQ, primers and the buffer
o RT – PCR Æ or reverse transcriptase PCR where rather than starting from the
DNA we start with the RNA, hence we see what is being transcribed in the tissue
of interest we what happens is that we produced the cDNA at reverse transcriptase
process which occurs in 37-50 C which takes 30 minutes and then we do the PCR
so this can be done in one step where we put in all the material of both the reverse
transcriptase and the PCR together and what happens is that since we have the
primers of the PCR already in then we will only reverse transcribe the segment we
are interested in and then it will go through the PCR to be amplified, while in a
two step process the reverse transcriptase will reverse transcribe all the sequences
then we will put it through PCR to amplify only the needed sequences
ƒ Note that these multiplied sequences can be loaded in a gel to see where
these genes where expressed, as oppose to the normal PCR which will
only multiply a sequences that is put in it
 ƒ RT-PCR is also called semi-quantitative PCR but the main question here
is that can it quantify how much a gene is expressed in one location and
not the other?
x For this to work we need to look at the relative amount of the
strands during the growth or multiplication phase which is referred
to as the exponential phase and not at the end of the process since
at the end we will lose the ability to discriminate between the
amounts so we need to know when the RT-PCR is going through
the exponential phase and take samples from there to see the
quantification difference
ƒ RT-PCR is also known for its low precision, low sensitivity and low
resolution where for us to be able to find or quantify a difference between
the values it should be of a 5-10 fold difference to be resolved on the gel
ƒ Hence we suggest that RT-PCR to be only used to see if expression is
happening and not how much
o REAL TIME PCR or REALT TIME RT-PCR Æ this is also referred to as the
quantities method because it tends to overcome the limitations of RT-PCR using
florescent dies that bind to the doubling sequences during the growth phase and
will quantify them directly and this will allow us to identify differences as small
as 2 fold in the material hence this will allow us to quantify the differential in
expression between the two locations or tissues
ƒ Quantification can be of 2 types:
x Absolute – is when we produce the actual number of sequences we
have in a certain tissues and what we do here is amplify a sequence
of the strand we are looking for, we form many dilutions of it then
apply it to fluorescence and form standardized curves to which we
compare our sample to and be able to produce how much
sequences we have but this is usually not what is commonly done
x Relative – where we relatively see which location or between
which two tissues we are producing more and by how many folds
and we usually compare them when they are normalized to a house
keeping gene that doesn’t change its expression during different
environments and in different tissues and this is what we are
usually interested in, what happens is we compare them to a
threshold which is set by the compute or the researched within the
exponential phase of growth and the faster we go above the
threshold in different tissues will reflect that we had more copies
initially that is why it took less time for it to reach the threshold
which is reflected in a lower number of generations to reach the
threshold and a lower CT value which is used in this procedure
 x So:
o RT-PCR Æ do it if we just want to know if a gene is expressed or not
o Real time PCR Æ do it if we want to know the level of expression, quantify it and
it is much faster to do due to the more sophisticated equipment which allows for
faster heating and cooling and it is more precise and accurate and more expensive

Micro-arrays:

x These will allow us to go from the gene to the whole genome where we have these
transcripts but we want to know where and how much are they expressed and we can do
this for many genes and not only for one or 10 like in PCR, we can also find which genes
are expressed in a certain situation and not the other and are certain genes only expressed
in certain cells and can be used to mark and identify cells
x We can also see which genes are being expressed in a fast growing fish but in our micro
array we are limited to only what we have transcribed previously and have them on our
DNA library that will allow us to make the micro array disks, so for example if in a
library we made transcripts of a healthy fish and hence if we are studying a certain
disease we will be limited while using micro array since we have transcripts only from
our healthy fish
x Note that the genomes are found in all cells but the trancriptomes are only found in the
cells of specific types, developing the micro array is a very labor intensive process
especially for fish species where it has not yet been developed for all , in a micro array
surface we have spots each holding a multiple copy of a gene sequences
x One we have the micro array disk what we do is:
o Collect the tissues of if desire of the two forms we have
o We put a solvent used to extract the RNA because we want to know what is
expressed
o We mix with a vortex then we isolate the mRNA after centrifugation by using a
bed tube and then we dilute them
o What makes micro array work is the fluorescent signal, we label each treatment
(healthy VS cancerous) with different colors and we do so by copying our mRNA
by a reverse transcriptase using fluorescent nucleotides
o The principle behind micro – arrays is hybridization so on the micro array we
have the single strands, so we put both labels samples on same micro array and
they will find their complement, if micro-array is not complete then some of our
strands will not see any complements to which they will bind to
o We wash the excess segments and scan them into a computer which has a grid
and tells us which is expressed and when was not
 o Yellow spots is where we have expression of both and by the level of
fluorescence we have we can identify how much of the expression has been made
o Genes expressed in one condition and not the other are said to be up regulated in
that condition
x keep in mind that the large number of up regulated genes seen in one situation can be a
problem in one of the transcriptional regulators up stream and may not be a mutation in
all genes, sometimes we have limitations in that although we have same level of
transcription of a certain gene we don’t have the same level of protein translation in both
conditions hence although they are both transcribed in both condition we can’t really
know if there is later an error in translation of the gene
x what is old about is that it is based on the old concept of complementation and what has
enhanced it greatly is the high density spotting robots and high efficiency scanners
x Micro array platforms can be of 2 types:
o Spotted arrays – made of a cDNA array produced from PCR followed by robotic
spotters and can be done by shorter oligo arrays
o In situ arrays – this is where you give the company the actual sequences and they
will build an array of it using no biological data, and it is more accurate but also
more expensive
x Experimental design coordination is essential for producing good data from micro arrays
where we need to think of the degree of freedoms we want in an experiment where the
higher it is the more statistical power we get and the degrees of freedom = number of
independent units or arrays conducted (-) the number of distinct treatments we have

DNA Sequencing:

x This might be used for a gene of interest or a population study will require sequencing at
some level, it started in 1977 by maxam-glibert were he used radioactive labeling but
then it was changed into the sanger chain method which has been used until the
development of next generation sequencers in 2005
x Sanger-chain sequencing – we have a primer where we are adding nucleotides with a
missing hydroxyl group which causes the chain to be cut and then we will run this
through a gel and we will be able to resolve the developed sequences
x Another method is the dye terminator sequences were we use fluorescent nucleotides
which can be read in the same way by stopping the sequences and hence develop the
required sequences from it
x Next generation sequencing, started in 2005 with 3 main platforms produced by solexa-
solid-roche developed through nano-technology
x This type of sequencing has reduced cost, work load and time needed to develop a certain
sequences and has broken the barriers for some species to go into the genomics field of
study
 o What limits it is that when we have or need to sequences a new genome that we
don’t have a reference genome to know where they are going to line up and may
require putting together many technologies o reach what we want
x Sanger still provides the longest read length for now and sanger still used for cloning
projects and form small scale production
x Micro arrays still have a high start up cost but still cheaper than next generation
techniques but next generation still provides information for transcripts and sequences we
have not yet transcribed and put on micro arrays discs and this provides a huge advantage
Î many consideration to which method to use such as scale of project, cost …

Linkage:

x It is physically the associated genetic loci, and this is why we have certain traits that go
together such as fast growth and a certain physical characteristics, the further the 2 loci
are from each other the less the chance that they are going to be transferred and the
higher the chance of a cross over event that will usually lead to genetic diversity
x This distance are amount of linkage between 2 loci is stated or given in centi Morgan’s
which reflects the percentage of recombination frequency, where the larger they are the
higher the possibility of recombination on the other hand independent assortment
involves recombination of greater than 50%
x How can this be used for a practical application where we use the genetic recombination
frequency to see how genes are located on a chromosome and if 2 traits are very close
and linked such as weight and disease if we choose for weight gain then we will also be
choosing for higher occurrence of disease
x Let us say we have 3 genes K,X & Y like in the example, we should note than the relative
in terms of percentage expression of each reflects different alleles, we have developed for
these genes genetic markers which has been developed using polymorphic repeating
sequences at the end of the sequence, we mated a male and females and took their 20
offspring’s and tested them for these traits and we did so by doing their genotypes, we
expect that each individual offspring to have the same genotype for all 3 genes, we look
at those that don’t and we take their percentage and that percentage is the centi Morgan
distance between the genes that are different in that individual so this is how we do it Æ
so we use certain software to develop the gene map on a certain chromosome
x Each of these genes that we have located is associated to a certain trait, also note that
within these offspring’s we can observe certain trends that we can go back to the their
genotypes and try to understand what is causing those trends
x The alleles A-B-C-D are basically values or how much of the genes we are studying to
reflect the genotype at hand
x When we use many markers and input them in these genetic systems we can develop
linkage maps of whole chromosomes and these markers can be of type 1 which are in real
genes or type 2 which are anonymous or non genes sequences
 x The LOD score is the logarithm of odds it is a statistics used for linkage analysis where it
will test the fit of these loci by associating them a LOD score a chance of this failing is
1/1000

QTL:

x Is the Quantitative trait loci the primary reason in aquaculture is to enable to identify
regions that are effecting quantitative traits which are of importance to us such as growth
and other which are also considered to be continuous traits that will help us develop
marker assisted selection
x QTL can be associated to genes that can directly affect or closely linked to it, it makes it
difficult to find that gene because it is closely linked to other, QTL assisted selection uses
QTL markers to identify or select for a trait characteristics hence it can be used to screen
the brood stock as a young age to be able to identify the needed sequences
x After identifying these sequences we do something called marker assisted intro-gression
where we cross the fish with the traits we want and result in the good fish of ideal traits
and the flounder is an example of this on the slides

Physical Maps:

x Is an actual map made if we line up all the ATCG sequences in a row this is the actual
map that we get, it is much finer mapping allowing us to select markers which are highly
correlated and linked and hence making QTL markers much more tightly linked
x It requires a big genomic DNA library and how we do it is we need to order the clones in
the correct sequence using fluorescent finger printing where we cleave our back clones
with these fluorescent restriction enzymes and we end up with fluorescent colored
cleaved segments and then the computer software will line them up and develop the
correct sequences
x So when we decide we want to develop a sequences that we want to amplify we go to the
physical map then to the clone that helped us develop that region which we can use to do
a marker or we can amplify and use it, note that linking our physical and DNA sequences
is done by utilizing micro-satellites
x It is essential for us to know the conversion between base pairs and centi Morgan’s and
we can do this by dividing the whole number of base pairs over the total length of the
chromosome in centi Morgan’s

Pseudo-linkage:

x Salmonids have always lead the way in terms of genetic tools used in aquaculture species
and this is why they went into a lot of trouble sometimes because they were leaders and
since salmonids are tetra-ploids (4N) and a lot of different occurrences occurred during
crossing over which lead to abnormal conditions (refer to note section in slides)
BACK TO SELECTION SECTION:
x If we reach this selection plateau or this high point it is the max gain with longer
selection but we can overcome this selection plateau because we need to exploited this
genetic variability hence if we relax selection we may lose some of the selection response
we got – because that hidden variability is there but is hard to detect – to break out of this
plateau we will sometime naturally brake the plateau when the hidden variability is
exposed due to recombination events that take place a few times after the plateau is
reached
x Logically there must be a limit where additive genes can induce an effect but some traits
such as body weight since they are affected by so much genes then it will maintain
enhancement over tens of generations
x Also with time we might have mutations that will vary the genetic information
o NOTE: this is an in-class idea – when we reach the plateau maybe if we select
very highly then we might lose these hidden variability hence it might be
beneficial to relax selection a bit
x Also bringing in new blood by cross breeding (called top crossing) to add this genetic
variation – if the brought in DNA is inferior we will depress growth a bit but due t
enhanced genetic diversity we will reach back a higher plateau, if we were able to
purchase an equally good or better strain then we will not get the depression and we
might have direct enhancement and even higher plateau
x When we are actually doing selection we sometimes have control over the selection
differential and sometimes we don’t, this is due to sometimes the conductions of our
production controlling what we do such as having limited facilities that can accommodate
a certain number of fish hence we should limit the intensity or sometimes we require a
certain number or amount of brood-stocks hence we need to select a certain number of
fish
x In selection we have been exposed so far to selecting the bigger fish or those that have
the advanced characteristics but this selection can be done in a positive way and a
negative way and when both are doing together then this is called a bi-directional
selection
x When we are doing this type of 2 way selection we will produce a heritability for each of
the directions and then get their average, this is usually used when we have an a-
symmetric response in a selection direction where the selection in one direction for the
same trait will have a much different heritability than selecting in another, such as
selecting for smaller fish is easier than selecting for larger ones, hence using both the
positive and negative values of heritability as an average will produce a value that is
more conservative than if we use only the positive direction that may sometimes be an
overestimate
 x The bidirectional cross we should note is not done for practical reasons but rather for
basic theoretical science values and hence not really used for our improvement programs
x Since response is different in each direction we would consider sometimes that each one
is a different trait that we are selecting for such as increasing WT is different than
decreasing WT
x In case of a chicken it is easier to produce or select for smaller eggs since basically they
can come out easier than larger eggs
x Very important Î if we have a moderate to high heritability we can use selection and if
low it is better to cross breed, BUT it is not as straight forward as this seems since if we
seek to form progression via selection we need to have high heritability coupled with
high variability for us to work with the hence if we have high heritability with low
variation we may improve by one unit but since we don’t have enough variation in these
additive genes then we might not get a lot of response while in situations of lower
heritability and higher variation we can get several units of change which may be much
more desirable
x Note: that at some point we will come to see that the best way to get the ideal genotype
we should use many genetic tools such as hybridization, cross breeding and other and
ultimately produce the best genotype
x The ideal situation is to have high heritability, a high level of phenotypic variation and
for us to select intensively since we should always remember the formula that:
o Progress = intensity of selection X variation of phenotype X heritability

Controls:

x In genetic experiments it is essential for us to have genetic controls and here in selection
they are very important, the most common is from each generation we have a randomly
bred control within each other and we compare it to the selected bred lines, the main
problem here is that due to environmental variations or our care of the fish the values that
we might produce tend or can vary greatly and hence change the control values a lot
such as when measuring the selection response as the average of the control parents
changes the value we get for the heritability may vary greatly
x We can attempt to correct these by possibly changing these values into percentages that
gives a heritability value that makes sense
x Another way to correct this method is by doing: Standardized selection differential where
we convert our data into standard deviation equivalents and work with those where we
for example have a standard deviation of 20 and the difference in the denominator of our
heritability equation is 30 grams, to get its standard deviation equivalent we divide it by
the value of the standard deviation to get a value of 1.5 and this is what we use
o This standardization method will produce lower quality values and numbers but it
can save our experiment
x Note that we can never use a previous control’s data for the next generation because each
grow out period varies in terms of environmental conditions and this is the basic reason
we use a control in a genetic experiment to be able to account for the environmental
component of the results we get
x Other options for us to use controls:
1. Random bred – discussed above, to even further enhance it we can increase he
population size to be able to generates much close to our initial population as we
can
2. We can re-breed the initial parents where in the next generation although we are
working with the progeny of these parents as the selection, we rebreed the initial
parents and get a new population to compare it with the products of the selection,
we have a problem here is that the original parents have a limited life span hence
in a long term experiment this is not very applicable in addition the parents on the
control will be of an older age that the parents of the selected progeny hence we
might have a maternal effect occurrence
3. The control of an experiment can be the previous generations selected line, where
here the control is not a bad performing control but rather a good one and we
would be comparing our new selections performance compared to the past
generations selection, this has more of a commercial application but in terms of
scientific research it is not a very sound method because the previous selected line
that we are comparing this generations’ offspring to is a result of the errors that
we are getting in our selection testing since each selection test has a certain
standard deviation and as we use these lines further and further we might be
compounding these errors over time
4. We can make also a standard cross breed, where they should somehow produce
standard fish but the disadvantage is that we have 2 maintain 2 lines for us to
cross each experiment and hence this will take care of the genetic drift which
might be a potential problem in random bred lines
5. The fifth possibility is for us to use homozygous clones hence we will produce the
exact same genes each generation but do have some problems which that they are
difficult to produce and those that are produced tend to have very low
reproductive success in addition since they are all homozygous clones then it
means their phenotypes are going to vary greatly with micro environments hence
they might not be a very good control at all
6. F1 clone cross bred – this might be the best control we can use, where we have 2
cloned homozygous lines and then cross it to produce F1 which are all
heterozygote but genetically, mitochondrial and cytoplasmically identical and
since they are heterozygote they can eliminate the effect of micro climates and
since they can be reproduced at every generation then we will have brood stock
for long terms experiments and have no maternal effect
 7. The last type of controls can be done using the production of fish by making 2
sets of homozygous lines for different genetic markers and we can’t fully produce
clones because we don’t have markers for all genes but we get as close as we can
to homozygous clones as possible

Different types of Selections:

x We have 2 basic types of selections which are individual or mass selection and family
selection
x Individual selection is where we choose individuals on their own singular performance
while in family selection we choose the individual based on his performance and those of
who are related to him such as by doing sibling analysis, parental analysis and others
x Advantages of mass selection is that it is easier to do because we don’t have to follow
pedigrees or families hence we choose based on individual performances and it is less
costly whiles its disadvantages are we have probability of choosing or selecting relatives
and tend to increase inbreeding which may eventually result in depression and it is not a
very accurate cross since we don’t follow records which in itself is increasing the risk of
inbreeding since we don’t follow pedigrees
x Advantages of family selection are many:
o It is based on more information on his relatives and makes it that our selection is
more accurate and allows us to choose an individuals that has a better breeding
value
o More records that will help s avoid inbreeding
o Allows selection for traits that usually cant be done on an individual’s fish such as
dressing weight, since if we do it on the individually selected fish that we caught
then we kill the fish and don’t have it anymore to reproduce, in addition we have
some traits which are linked to a sex hence for example if we individually
selected a male we cant evaluate him on egg production because he doesn’t
produce eggs but in family selection we can evaluate his sisters and relatives to
get the value of this trait
x The advantage of individual selection is its simplicity and lack of facility requirements
and less cost as oppose to the family selection another advantage of family selection,
although when additive variation is decrease and hence the heritability drops or is masked
by the environment, family selection will allow us to shed away this environmental
component have allows us for doing the progress – choosing families in some
mathematical way allows us to see the differences between environments and hence
allows choosing based on additive variation
x There are many types of selection and these are some examples:
o With egg production is chickens they reached a plateau where they produced 2
eggs every 24 hours, hence scientists manipulated the photoperiod in a closed
environment to produce 2 eggs every 22 hours and this enhancement was shown
 as enhanced genetic gain because when we reverted to normal light hours the
chicken stayed producing eggs at the 22 hour rate
o Another example is that there are some traits which are controlled by major loci
such as the number of hands or limbs someone has but we suppose that there are
many loci that support this main or major loci so that if the major loci failed the
minor ones will ensure that the normal trait is produced
o Another example of this major loci concept is found in flies that normally has 6
bristles, where although people tried to change that it will not change because it
was a major trait controlled by both major and minor loci (like how important our
number of limbs are) but by shocking the eggs with a cold shock we observed
variations in genetics where the average number of bristles on a population level
increased and when the temperature was reverted to normal then this genetic gain
was maintained hence the genetic makeup was maintained
o In channel catfish for example, 3 generations experiment showed an increase in
BW where the top 10% were chosen, the 1st generation showed progression,
followed by regression in the 2nd and progression in the third the difference of
growth improvement was also observed between the different lines of Kansas and
manor catfish
x The vary rapid gain in the 1st generations of a selection program are very typical but this
is where also that the continuation of the genetic gain will stop at some point – the cause
of this is due to epistatis and dominance can effect early selection response where
although the heterosis or advancement we see are due to additive genes but we do have
some portion of these trait transferred with dominance and this is what causes the
advancement early on until the population as they say is FIXED and no more dominant
effect is seen and the growth will go back and increase but at a slower rate which is
characteristics of only additive gene components
x This is also seen in the Kansas catfish example where we see a fairly good rate of
increase at a value of around 55% in the first 4-5 generations but it eventually slows
down
x When we are doing a multi-generation experiment at every generation cross we can
calculate a value for the heritability that we can measure at each stage and this will allow
us to eventually measure the cumulative heritably after a longer period of time has passes
(the value will be more of an average, and will reflect lower value than the previous few
generations because the 1st and 2nd generations are usually highly inflated) – note that the
cumulative is usually low since when calculating it in the denominator we are adding the
selection differentials of all the selections and this tends to inflate the denominator and
hence reduce the cumulative heritability that we can eventually get
x Selection for body weight works more than 90% of the time, hence selection is a good
way to increase that characteristics
x In fish the better the strain we start with our selection program the better fish we are
going to get as a result, hence having the ability to choose our strain we have the
capability of instantly improving our traits, when we look at the response which are made
conservatively since we will have some inbreeding effect in our select line as oppose to
random breeding in the other lines hence the amount of enhancement we see is somewhat
conservative since or due to this inbreeding effect in our select line
o Such as in our example of domestic strains growing at 2-6% per generation, this is
conservative since we have some inbreeding and hence the results are more
beneficial in our control lines and this is good so that we will have practical value
for us to tell the farmers
x Many fish tests were done on many species such as ROHO fish showed improvement, 4
generations of blunt-nose sea bream showed 19% improvement in china, silver barb in
Bangladesh observed different strains reacted differently to selection, in Israel they found
out that heritability in tilapia for BW in selection was zero while in different or within
some families it was much higher at 5-10%, oysters also showed an improvement
x In Tilapia, mass selection increased body weight by 15% in Oreochromis and
Mossambicus but Oreochromis showed more impressive results since it achieved this
enhancement in less time, noting that results here are questionable since inbred lines were
used
x Data on slide 244 was for oreochromis were we see the value for the damn and sire
heritability values of tilapia growth at 45 and 90 days, where we see that sire heritability
was close to zero at both dates while the damn it was high initially and dropped and this
is because of the maternal effect which is inflated in dam heritability but tends to
decrease with time and hence the trait has generally low heritability as observed
o The data on slide 245 showed that the actual realized heritability did prove that it
is actually 0
x At this point we see that Tilpaia is an exception to the role when talking about selection
to increase body weight since it showed no heritability but the GIFT project showed an
increase in weight
x Usually in all fish we typically get 7% gain of BW per generation due to a 10% intensity
selection each generation but for catfish, Atlantic salmon and GIFT tilapia we were
getting 10-15%
x Why did the GIFT project show positive results as oppose to others Î due to wide base
of original genes where each different researcher worked on a specific strain the GIFT
project developed a base of fish developed from 8 strains which were intentionally cross
bred to enhance the genetic variation
o These 8 strains where from both Africa and within the Philippine / the Israel,
Singapore and Taiwan strains are Ghana based and the Thailand is Egyptian in
origin
 o What they found out surprisingly is that the wild African strain grew more rapidly
than domesticated Asian but can be explained (noting that Asian brought in first
the mossambicus strain when it turned out to be very bad in growth they switched
to Nile tilapia and it seems that since both these fish were out of their natural
environment, they had a high hybridization ability so the populations where
contaminated) so this reflected in Nile tilapia with low growth due to genetic
degradation since the wild ones were contaminated, other potential effects where
the inbreeding since the initial populations were brought in small sized groups
that bred together hence may have depressed the population, and random genetic
drift could have also contributed to this problem as well
o GIFT project – after developing the 64 lines they chose the top 25 performers and
worked on those / but what they should have done to make sure that they are not
getting rid of a good trait that might have been masked in the F1 they should have
produced the F2 to ensure that all alleles are mixed well together and the genetic
variation that is produced there is much higher
o The result of the 1st 7 generations of the gift are given in a table in the slides, with
the normal high enhancement initially and then reverting to the 6-7% growth
which is expected (note that normal or expected heritability for BW is 0.2-0.3 per
generation)
o The Gift program also had an advantage over others is that it didn’t only use mass
selection like the others have used but it also used all 3 systems of mass selection,
family selection and indexing (pedigree following)
ƒ NOTE: when we discussed the different types of selection being mass or
individual, we need to know that the highest or most successful type is
when we mix them both together where we or choose individuals within
groups based on following their pedigrees
x The gift program may not be as successful as it looks where although in all selection
programs people seek to increase gain of good alleles and decrease bad alleles what the
gift program has been shown to do is basically working to decrease bad alleles only that
they have introduced into the population to start with initially when we made our base
population with the 8 lines – we observe that as we progressed the Asian component of
the selected lines have been decreasing every time, noting that at the 7th generation, 75%
of the line is African mainly Egypt-Kenya-Senegal strains and the rest was Asian noting
that of the left 25% - 24% was Thai which is of Egyptian origin Æ hence we see that we
are basically removing the Ghana and the Mossambicus lines since they are the worst
performers
x Hence the experiment or work can be seen as removing the bad performers Æ so in retro-
spec it might have been good to start the mixed population with only the better African
strains so that we will not loose several generals in the selection process removing the
 Asian bad strains (of Ghana and Mossibicus origin) which we knew are inferior and we
knew that the Asian strains are contaminated with them
x Slide 258 shows a 6 generation selection of 2 lines of rainbow trout which produced
around 30% enhancement over that period of time with Atlantic salmon showing around
100% improvement in 10 generation hence we can safely say that body weight
enhancement in selection varies between 5-14% per generation and the mean is around
7%
x Some practical things about selection, if we have several ponds in which we have fish
and we want to select larger fish from different ponds we should not pool all the fish
together and then choose the bigger ones but rather we have to choose the bigger ones
from every pond and then combine them together, since the phenotypes in the different
ponds for the larger fish in each pond will be different due to the different environmental
components hence if we pool then select we might only be selecting the fish of one line
and not the other, hence we might lose some good genes that we might have had
x In some species we have sexual dimorphism where the males and females grow at a
different rate such as in tilapia males are faster while in salmonids females are larger and
grow faster, hence if we do a selection at an age where we can’t sex the fish such as in
fingerling stage by grading them we might catch all the fish of one sex and not the other
hence in the population that we are going to make up we can’t get them to reproduce
o Hence when we select we should be sure to get both sexes and this sexing can be
done using a pencil technique or a blunt needle to see that the females have the
larger hole in which this pointed instruments go into to
o Note Æ if we choose to select only for one of the sexes and then use any size of
the other to develop our population what we will be doing is actually reducing our
selection intensity where we will get a response but not as much as what we
would have gotten if we selected for both
o For sexing some blue dyes can be used (such as in tilapia) to help with the sexing
technique at small age
x Several reproductive traits in salmonids have been improved due to their high rate of
heritability and this is a general rule where in general most reproductive traits can be
selected for due to their high heritability in most fish such as age of sexual maturity and
for time of spawning where for example sexual maturity in Atlantic salmon has been
reduced from 5 to 2 years in salmon due to this
x Heritability for other traits have been measured and tend to vary greatly based on the type
of trait, the species of fish we have where for example:
o Dressing percent had a heritability of 0 while for fat percentage it had a
heritability of 0.6 (the case with fat is that when we choose for larger fish size, it
will increase the fat content because they are highly correlated)
ƒ Note on measuring increased meat production, it is Nonsense to measure
the size of the fillet since simply larger fish will have larger filets, and
 based on the body shape the dressing percent will change hence
standardization with a one or two fold regression is essential (it is very
important in any paper to define what we mean by dressing weight)
o Body shape has also been seen to be highly heritable with a heritability in carp
ranging from 0.4-0.8
o Heritability values were also developed to increase body weight in Australian
swap crawfish which basically refer to the tail meat
x regarding disease resistance, selection against bacterial and viral diseases showed that 50-
70% of the selected lines against such diseases showed improvement of varying levels
but this is not successful as accuracy of BW selection which is at a solid 90% that we can
achieve
o in catfish, selection for bacterial disease were successful since both sire and dam
heritability were not equal to zero but for viral disease sire heritability reflected a
value of 0 which means it is most probably not heritable, but note than in both
cases female or dam heritability was many fold larger than that of the dam, hence
it is clear we have a maternal effect that is giving these fish an advantage, so if we
clearly pinpoint it out as being a genetic component that we can select for or
something environmental we can adapt our management techniques to fit it
x The table which shows different strains or line and measuring the heritability of diseases
we observe that their values tend to vary greatly between line, where for example
heritability of resistance against colomnaris varied between -0.03 to 1.13, hence in the
cases closer to 1 we can say selection was successful
x Since apparently we can say that little additive genetic variation can effect selection in
some situations and in some diseases
x Also resistance to nitrite poising can be done as well as oxygen tolerance with varied
results based on strains, where we have success of choosing other traits of 50-60% in the
case of catfish for example:
o 3 of 5 selected lines respond to low DO resistance
o 2 of 3 to high NO2
o 1 of 3 resistance to ESC
o 2 of 3 resistance to colomnurais
x We can somewhat safely say that cross breeding enhances disease resistance more than
selection but this may results in us risking other traits being decreased and hence we
should be very careful for this by not taking our eye of the other traits which may be
interest to us
x The merestic trait herbalist is very high, but in most cases this will relatively have low
phenotypic variation and in practical sense we will not need to do anything with in for
commercial producer because we don’t really care if we have more scales or less, in slide
276 shows the bi-directional selection for dorsal fin size in 3-4 generations and we get a
 differential results since in some cases (such as this) decreasing the fin size was easier
than increasing it

Genetic correlation:

x Or the correlated response to selection, where different traits can be generally correlated
in 3 different levels:
o Positive – where if 2 traits were linked, when we increase one the other will
increase
o Negative – where if we increase one trait the other will drop
o Zero – the traits are not effecting each other
x To get a positive or a negative correlation we can do genetic correlation tests or values
for sire, dams or mixed where we see that the equations contain both traits and values for
them being X & Y
x How we can use the correlation if we select for one trait it may have a negative, positive
or zero correlation on another trait hence if we select and have an effect we can predict
the correlated response of the second trait Y based on the selection of X
o Cry = i.¥K[¥K\U$3
ƒ Where Cry is the correlated response of trait Y, I is the selection intensity
and Hx & Hy is the heritability of each trait and rA is the additive genetic
correlation and P is the phenotypic standard deviation
x So the Hx & Hy we can density it from previous experiments that we can conduct and
then plug them into the equation if we factor this equation it will tell us which type of
selection is more effective since there is a possibility of indirect selection what we are
doing in this type of selection is although we seek to increase one trait we tend to select
for another which is highly correlated to it and in many cases we get a very good
response the formula is:
o CR x / CR y = rA . Iy/Ix. ¥K\¥K[ DQG LI WKLV UDWLR LV ODUJHU WKDQ RQH WKHQ ZH
should do indirect selection if it equal to one then both methods are equally as
good and if it is less than one than direction selection should be done
o In this equation varying the intensity of selection for Y or X may have an effect
on the value of the ratio and hence may help us choose one selection method over
the other
x An example of this indirect selection is that if we want to improve FC we see that another
trait like body weight is highly correlated to it so we select for that since it will be hard to
quantify Fc at each selection stage, another reason for choosing indirect selection is that
sometimes we will need to sacrifice the animal to know the trait like dress out percentage
hence finding a correlated trait to it such as body density which is highly correlated to it
is very effective - note that body density in catfish is correlated to dress out percentage
while catfish morphology is not
x The work on indirect selection has not been done a lot but some correlated responses has
been done, example in red swamp crawfish where if we select for late sexual maturity we
would not necessary choose or get faster growth rate but rather we will get a larger final
size
x If any genetic tool such as selection, cross breeding is used, we should note that faster
growing genotypes is due to improved FC and increased appetite and both are need to
contribute to better growth
x In channel catfish fecundity was positively correlated to choosing catfish of larger size,
but there is no correlation between body weight and body composition in channel catfish
x An example of negative correlation is seen when we choose for larger body weight the
fish became less tolerant to low oxygen where it took the selected fish ½½ the time to go
into stress than the randomly selected strain, noting that the tested fish from each group is
of the same size hence we see that their inability to pick up the oxygen is physiologically
genetically linked and maybe be due to higher or lower inherit metabolism rates
x An example of a positive correlation is in the marron where the dress out percentage in
selected lines is 1-1.5% more than randomly selected lines
x Genetic correlation to resistance for various diseases where we see that we have different
correlation between different diseases, the significance is that when we talked about
heritability the channel catfish virus has a zero heritability but since we have a correlation
with other diseases we can decreases the virus incidence by indirect selection to the
diseases it is correlated to
x When we look at growth rates when we select for food fish size there are many other
traits which are correlated to it such as fingerling size and higher consumption, and it has
been seen that younger fish that are of larger size is correlated to larger fish at an older
age
x Slide 290-291 shows that after 6 generation we see a negative correlation between it the
disease and body size
x In the end of this study we have a positive correlation between survival and some other
traits such as:
o 1 of 1 of those selected for low DO showed better survival
o 2 of 3 chosen for high NO2 tolerance
o 1 of 5 chosen for Colomnaris resistance
o 2 of 3 chosen for ESC resistance
o Î the lower these values it tells us that using this trait for indirect selection is not
going to be a good idea
x Slide 296 – CXC means it is random and MS means selected for body weight, we have a
negative response to edwadsialla disease where controls are more resistance as oppose to
Kansas select (KS) and Kansas random (KR) is dyeing more quickly than the Kansas
select hence we are having different results in each cases – it tells us that the genetic
 makeup that effects correlations and heritability in each population is different and hence
no one value can be standardized on the whole population
x Slide 297 – is done on or for colomnuris noting that CXA means it is a control, this
shows us that the 50% mortality and the time to total mortality should be treated as
different traits because they give us different results and are not consistent with each
other
x Slide 298 – continues to discuss how these different selections relate to parasite species
showing that Kansas selected for Body weight had 3 times the amount of parasites than
the randomly mated Kansas and hence the traits are negatively correlated
x Slide 299 – we have selection of different traits and see how much of a percentage gain
or effect on correlation we have on colomnaris, it appears that all survival traits we are
selecting for has a negative effect on collumnaris occurrence
x We seek to use this information in management applications hence rather for trying to
select for resistance fish we might need to manage better the grow out system since not
everything can be corrected for by genetics
x The overall conclusion is that selection or indirect selection for water quality and diseases
had similar results (refer to statement on slides)

Disease in Salmonids:

x Selection improved disease resistance in salmonids have been successful but in some
situations we has negative correlation such as in the case of Sockeye diseases, where
when we chose to make one disease better we pushed another one worse and Vice versa,
hence for us to know which disease we want to genetically control we either should look
at which disease has more impact on our production or see if w can reach a mid level or
midpoint between them or even see if we can reduce each using different tools maybe
one genetically and the other by different environmental or management measures
x Slide 308 – shows that although 2 traits might be phenotypic ally correlated does not
always mean that they also have a high genetic correlation
x In the case of tilapia, late sexual maturity has a high heritability, but early sexual
maturity is something that we really don’t want since it increases our fish density and
hence reducing growth rate, but late sexual maturity is strong negatively correlated to
growth rate, hence choosing for late maturing fish may solve the problem of the
population but may result in very low growth rate
x What the Norwegians suggested although choosing for both traits might seem, not to
have a good result, but long term selection for both we might improve them both because
there is a chance for recombination events to happen that will allow both traits to be now
positively correlated
x Multiple trait or tandem selection is when we choose for one trait for some while then we
choose for another trait for another while, the problem here is that we gain initially might
be lost and VV, another way to do this is by developing a minimum cull level system for
 both traits where if an individual doesn’t meet this minimum level for any of the 2 traits
we have then we will cull it, the problem is that some of them may be very good in one
but not in the other hence we might be losing good genes
x The situation with GIFT tilapia as previously discussed, that it had 70% increased growth
but showed only 16.6% gain in genetics with increased seinbality, less cold resistance …
x Slide 312 – shows the correlated response of survival and in body weight where as we
are selecting body weight we are increasing body weight – this increase initially may be
due to cross breeding effect and dominant gene effect may be due to it and the loss we
see with time we should state is not due to inbreeding since in GIFT they were following
pedigrees to prevent this

Tandem Selection:

x This section has been previously discussed with the advantages and disadvantages of the
tandem selection and the independent culling level

Selection index:

x Is the best way for us to do multiple selection of traits, there are many ways to use it,
sometimes we might be interested in one trait and hence use this to develop an index for
one straight were we take the value of that trait at many ages as well of those of the
individuals who are relatives (this would be considered to be the family component) for
him to develop an index for that individual for that individual trait
x The index can also be determined for several traits and not only one and link it to its
economic value, hence if we are selecting an index to make multiple selection we need to
know and these traits are 4:
o Economic value of each trait since we need to develop using these different traits
a regression value which can be considered a weighing value for each of these
traits, hence the economic component will reflect how much a trait has an
influence on our profit margins, hence some genes are more important than others
due to their economic component
o We need to indentify the heritability for each trait due to additive genetic
variation
o We need to know the genetic correlation between the traits
o Phenotypic variation and correlation between these phenotypes also needs to be
identified
x So each individual will have an index or breeding value denoted I where:
 o I = b1X1 + b2X2 + … + bnXn where b is the regression coefficient based on the 4
traits we have discussed
o This index is assigned for each individual in a population to decide on which one
we will select, because we will look at the composite value of the end and choose
based on that composite value
x Slide 320 – shows you that if we select for 1 trait (reflected in 1 phenotype which we are
interested in) and we want to choose the top 10 percentile then the number of individuals
saved or selected is much more than if we want to choose the top 10 percentile for many
traits and cull the others, this is what would happen in independent cull values where all
traits are given equal value unlike in this index concept where since we give a different
value for each trait then each one will have a different percentile value that we are
interested in, preventing the restriction of the population we choose
x Breeding values – is the value of an individual compared to the mean value of its progeny
determines its breeding value since genes are passed on with reproduction and not
genotypes
x The average effect of the parents in the progeny determines the mean value of its progeny
x The breeding value is only valid in the population we are studying now hence a breeding
value for same lined in different conditions is not valid because it compares the value of a
certain individual to the population he was in and not to others
x Breeding value equals the sum of average alleles of each gene input made over the pair of
alleles at each locus of our interest over all loci
x Advantages of using breeding indexes is that they indentify individuals with valuable
heritable traits allowing us to increase selection power and can allow for multiple trait
selection but can also lead to culling individuals with important genetic contribution (but
note that when compared to independent culling method this method relatively decreases
the probability of us eliminating good genes but tends to potentially be a problem since it
can remove good genes for a low priority trait that has a low regression index between
our different traits) and may apply sometimes too much selection pressure due to the
previously discussed problem
x The index is a composite of several characteristics based on the economic values
mentioned before and this is due to the mathematical relationship of matrices that we use
to measure or calculate this regression method
x Best program that was developed for this regression is the BLUP where we seek to
develop these indices with least bias, note that the method of measuring indices has
changed 3 times: (listed in order)
o BLP – best linear predictor – 1st developed and then improved
o BLUE – best linear unbiased estimator
o BLUP – best linear unbiased predictor – current method used and is strongest
statistically
 x These methods are evolving to enhance the accuracy especially that they are based on
averages that have a standard deviation that we seek to reduce in value as much as
possible
x If we are using these breading values for many traits we call it a multi-variate trait and if
used for one then it is called uni-variate trait
x We can develop a selection index to determine even one selection as mentioned early
one, since we are sending into our gametes to our offspring’s our genes and not our
whole genotype and the value of the individuals performance does not equal the value to
each one of his gametes but rather the total average of all gametes equals his performance
but each one of his progeny I sonly getting one of those gametes and hence a selection
index used for one trait is needed to help us better select and this one trait index can be
developed from family information, or from the same individual’s trait of interest on
different durations and occasion and this index can also contain different traits although
we are not interested in them
o Note that this uni-variate indexes can be developed by using also the information
of offspring if that individual has already mater to assess the quality of the
gametes he is producing
x In multi-variate selection the average individual will have a value of 1 and those of better
value will have a value higher than it and those lower will have a value lower than it and
we can use those to select
x The more records we have the more accurate our assessment of an individual’s value is
x We have advantage in fish since we have a large population hence we can ask a selection
process to select for many traits at different levels or intensities and still get a population
that fit this criteria which might not happen in other conditions

Polyploidy:

x Most but not all fish are diploids meaning that they have 2 copies of their chromosomes
some fish are genetically plastic and can produce triploid fish and we can sometimes
induce tetra-ploidy to occur and for some reasons that we will discuss later we want this
to happens, and the processes to produce those are similar to gynogenesis and
androgensisis
x Where to produce a triploid Æwe allow normal sperm to enter, we block 2nd meiotic
division to prevent polar body from being released and hence we have 3 sets of
chromosomes, one males and 2 from female
x Where to produce a tetraploid Æ here we allow normal sperm to enter, we allow the 2nd
polar body to be released and we get a normal diploid cell like always but we prevent 1st
cell division from occurring and hence produce a tatra-ploid
x We can do these chocks by several method either temperature, chemicals such as
Cytokalysin B (which disrupts the spindle fiber during division) or hydrostatic pressure
which prevents polar body extrusion by applying pressure on the eggs, and hydrostatic
 pressure is considered to be the most or best technique because the results are consistent
from one time to the next but for some types of fish other methods are much better such
as temperature shock for salmonids
x The time if when these shocks are applied is essential, because the time after fertilization
(or activation in case of irradiated sperm) that we need to apply the shock varies between
species, where the slower the development of the fish the later we can do the shock and
vice versa, where in carp the 2nd polar body is released 1-2 minutes after fertilization
while in cold water species like trout it can take up to 45 minutes
x What we need to identify for these chocks is 3 basic components:
o Starting time of shock
o Magnitude or strength of shock (such as amount of pressure we need to reach)
o Duration of shock period
x The original way for us to determine what time we need to chock and what levels was all
based on trial and error but with more research we are getting some better ranges for us to
start with that will reduce the amount of work to be done where it is found out now that a
pressure of 6000-7000 psi is good for fish but we need to say that it varies between
species
x The concept of TAU was developed where TAU is the ratio of the minutes to shock over
the minutes until first cell division, and for example in channel catfish time to shock is 5
minutes after fertilization and time to cell division is 90 minutes hence TAU = 5/90 and
hence we found out that a good range of TAU to be used as a good starting point is 0.05-
0.07, but this value changes with temperature since at lower temperatures everything
slow down and for different species as well, hence if we know when the first cell division
can occur then we use TAU to get as close to it as possible
x Note on pressure chamber use safety, it is essential for the pressure chamber to be
completely void of air when use and hence the utilization of bleed valve is essential,
because if air pockets are found in the water in the pressure chamber a potential bomb
can be used, and we can know when all air is out when the bleeding valve is releasing
water
x We are interested in triploids for many reasons one of which is that They produce sterile
fish that can’t produce any offspring’s
x Some scientists initially thought that since any cell naturally will tend to produce a
constant cytoplasm / nucleus ratio they thought that by producing triploids, that increase
the nucleus size by 50% they will increase the size of the cytoplasm by a similar ration
and hence this will lead to larger tissues and a larger animal but that was not true since
the animal can detect if there is large cells and will compensate by producing less number
of them
x In case of fish with triploid it guarantees 100% sterility and there is a very rare conditions
where triploid females were able to produce a few progeny causing some states banning
 triploid uses, noting that some states allowing the use of invasive species if they are
triploids because they can’t be used
x But note that in these abnormal situations the fish that created the few number of progeny
did so by artificial spawning and not naturally, in case of shellfish it is a bit different
because shellfish are genetically weird or different, where in most cases triploid shellfish
were completely sterile but in case of pacific oysters there is a potential problem where
there are shellfish processes that produce gametes differently where in fish you can only
get poly-ploidy by blocking the 2nd meiotic division but in shellfish you can either block
the first or the second because the eggs are ovulated before the first division occurs
unlike in fish and hence this produces many cases in which we can produce poly-ploidy
hence we can produce more than tetraploids so genetically shell fish are very different
and it has been documented that some triploid in fish is not permanent and may cause so
sort of reversion to the diploid form and hence may become fertile again
x Doing manipulations of triploid shrimp is done but genetic manipulations to produce
triploid or even genetic engineering in shrimp has been very hard to do since fertilization
in shrimp is internal and we cant access these eggs unless after being fertilized while in
fish they are externally fertilized so we can change the characteristics of the eggs before
them being fertilized but in shrimp It has been developed and triploid sterile shrimp has
been developed
x Triploid is the best genetic tools to induce sterility in aquatic organisms but it doesn’t
always disrupt sexual behavior
x In case of males even though they are sterile and don’t produce viable sperm they will
produce secondary sexual characteristics and look like male diploids (noting that in
triploids the gonads don’t develop well but all other characteristics do develop_ and if sex
hormone profile is looked at they will be identical to diploid males and since they have
normal sex hormones profile they will have same sexual behavior
x In case of females, knowing that normal diploids female peak at sexual duration
(hormonally) but triploids don’t have the same hormone profile because triploids tend to
disrupt the hormone surge which are associated with sexual maturity in females hence no
sexual behavior is achieved, these have implications on management where females due
to that lack of hormones they didn’t migrate (in case of triploid trout) where at sexual
maturity they should go up a stream hence we can use triploid females brook trout for
marinating a trout population in a lake for recreational fishing purposes
x Other implications is that males will also try to mate but he has no sperms produced so
they will fails and this also occurs when diploid females used with a triploid male
x When we have interest in a strain which we don’t want to affect the already present
population in a natural environment we can use triploids, the strain on interest it added
but doesn’t reproduce, in aquaculture environment where fish are well tended for,
triploids and diploids tend to have the same survival levels but triploids tend to have less
 survival in the wild and this is a very distinctive GXE interaction where the same
genotype is reflecting different growth and other traits when the environment is changed
x These results tend to also be found in wild and domesticated diploid (noting that results
vary greatly between lines and families used) where the better the line used to develop
the diploid the better that triploid is
x In the recreational fish triploid issue, potential problems is that the stocked triploids will
have worse mortality but this can slightly be fixed by stocking more fish or we can use a
better strain to make the triploid initially
x The reason they are having worse mortality in the wild is due to triploids being less
aggressive and this may be due to less hormones produced in both males and females
(females more so than males) and this will directly affect their feeding vigor where this
will lead them to getting less feed due to the diploid fish in the pond or natural setting
have a higher head-to-head competitive ability hence triploids show smaller size but
when diploids and triploids are separate these differences is not seen Æ this is again a
very good example of GXE inter-action, experimental results of testing this theory gave
opposite results in lab and natural pond condition since in lab the water was clear and
feed was sinking so the triploid fish had to put no effort in getting to the food which is
not the case in natural environment hence there food is much harder to get to and since
diploids are more competitive they will get to it faster and better
x The issue with triploids and getting larger fish has been mentioned where larger DNA is
compensated by larger cells but fewer in number, it is not well studied but having larger
cells size has metabolic effect consequences where as size increases the surface area to
volume ration drops and since the surface across all reactions occur which are important
to the fish we have less surface to do those (material exchange and others …) and within
the cell we have longer distances for the material to cross to reach target organics making
them less metabolically efficient for these reactions
o This have been seen to show that triploid salmonids and other fish tend to have
reduced tolerance to low DO and maybe linked to the issue of decreasing ability
to transfer material across the membrane
x Effect on growth rate is that vast majority of fish studied shows no differences between
triploids and diploids regarding their growth rate, although some conditions showed
di0ploids being better or triploids, triploid is not considered to be a good method or
strategy to increase growth rate
x But where growth rate is effected is after the point of sexual maturity is reached since at
that point a lot of energy Is naturally invested in developing the gonads and is shunt away
from growth hence we see a reduced growth rate, but in the situation of triploids where
the hormones are effected to a degree in both females and males, less energy will go into
sexual dimorphic characteristics and more will stay in growth of the body tissues – it is
essential to note that most aquaculture fish are not grown past sexual maturity hence
using triploids would not be very helpful there, but in certain markets that demand larger
 fish that can only be achieved after sexual maturity is reached then triploid can be very
beneficial such as in trout industry in Europe
o For example in tilapia since they tend to sexually mature so early, utilizing
triploid can be very beneficial but it is has been documented that triploid fry are
very hard to produce commercially
o In shell fish since they also mature very quickly, triploids is a very good
technique to allow for lager growth, note here that a huge GXE interaction takes
place for shellfish since they are filter feeders and hence as the environment
changes in terms of food available to be filtered out the growth changes
x On this note we have to mention that 2 main factors that tend to affect all GXE
interactions and changes:
o Probability of seeing a wide GXE interaction increases as the genotype
differences change or are further apart, and since diploids and triploids are very
different then the differences they imply on GXE interactions are wide
o Probability of seeing a wide GXE interaction are also further and wider apart as
the differences between the environment increases and becomes wider
x NOTE: triploids don’t override the sexual dimorphism in growth in some fish species,
where triploid male tilapia will still grow bigger than triploid female tilapia, so when we
see which method to use in getting bigger fish either diploidy or sex reversal
(development of mono sex population with hormones or XY-WZ systems) we should see
what purpose we are using them for, because mono-sex populations are still fertile, since
if they are let out into the natural environment they can mate with the wild population and
effect gene frequencies, in addition in producing these mono sex individuals a fertile
brood stock is essential to produce the individuals which we will sexually reverse them so
if these escaped this is a problem as well
x Using triploids in a commercial setting is beneficial to protect the germa-plasm we have
of our initial brood stock and the work we have done
x One more reason why triploids are used is that they can enhance flesh quality where
when males and females go into sexual maturity due to the hormonal stimulation the
body composition changes making their body protein less palatable, hence triploid since
it disrupts the hormonal indications in the body it tends to effect the flesh quality directly,
this is especially relevant when fish are growth to sexual maturity or a date close to it
(markets where larger fish are demanded) this problem is greatly facilitated by triploids
x Triploid is very important in oysters regarding their flesh quality where oysters tend to
have a large gonad compared to their body, and this gonad tends to be less palatable so as
long as we can work to reduce its size it is better and hence controlling hormones to
reduce its size and prevent it from getting bigger is very important and hence triploids are
very important to flesh quality
x Note: w way to produce triploids 3N is by mating tetra ploids 4N with diploids 2N, where
producing 4N is the problem because they tend to have high mortality and the cells that
 survive are very weak, note that if they survived 4N embryos are fertile – in some fish
this is more feasible such as in trout because they have come from a 4N ancestor and
hence more genetically receptive for being 4N
x Continuing with tetra-ploids the natural way to make 3N is by crossing 4N X 2N and not
done often because it is hard to make 4N due to their viability problem in one situation
was in trout were a male 4N X female 2N produced 96% triploid and the remaining 4%
are aneuploids and these are individuals which have incomplete chromosome sets such as
if we have N=20 and a triploid is 60 then an aneuploid might be 58, 59 or others and
hence has at least one incomplete set but since aneuploids are also sterile it is not a
problem but their viability is very low
x When we make a 4N, there is different timing of the shock where it can be a meitotic or
mitotic shock so it can be at the 1st cell division or even before or earlier where if we
shock earlier we are stopping Karyo-kinesis which is division of chromosomes while if
we shock later we might be stopping cytokenesis which is blocking cell division which is
the more common form
x Sometimes these procedures can result in mosaic individuals (where each cell of the
individual has a potential different ploidy number) hence we should always test more
than one cell for ploidy level due to this problem

Methods of determining ploidy number:

1. The best way is to make a karyotype by doing this chromosome spread and count the
chromosomes to develop the chromosome number and pairing them together, this
procedure involves breaking the cells and spreading them, stain and observe under the
microscope and we should do this procedure for many cells, the advantage of karyotype
is that it is absolutely definitive and the best but it is very slow and tedious process and
has a difficulty in staining and it is lethal to the fish because we need to sacrifice it
2. Another accurate method which is a bit faster that karyotype is the nucleolus organizer
regions (which is especially useful is smaller fish which are dividing faster) where a
silver staining technique is used when chromosomes condense and hence a 2N individual
will not have more than 2 of these organizers in a cell, while a 3N will not have more
than 3 and a 4N will not have more than 4 so we can see the max number of sets we can
fins of those and determine ploidy
3. A combination technique of NOR (number 2) and the karyotype can be done which gives
us results on both methods and the staining procedure to do so is much easier
4. The next method or technique involves making slides with cells where we look at the size
of the cell with a control and measure the other individuals (since as chromosome number
increase the cell and cytoplasm increase as well) and see by what proportion they are
larger to determine ploidy and measurements are done using micro-meters
5. Next level involves also the relation of the size where we use a coulter counter where we
want a quick method to test big shipments of fish where laws prevent 2N or 3N from
 entering hence this method can analyze as much as 1 or 2 fish per minute, were we prick
the animal take a blood sample and suspend it is a solution and then put it in a machine
that will measure the cell size and give the distribution and compare it to the standard
diploid that we use another nice thing is that we can use this procedure on embryos and
run them through the experiment where if we thought we have a problem in making the
triploids we can identify it early on. Although very fast this procedure is not 100%
accurate all the time but the mistakes are done are conservative since the error that it
might do is mistake a 2N for a 3N which is good, the machine is expensive
6. The next technique is very rapid as well and uses a flow cytometer which measures many
cells from an individual and averages them out in terms of their DNA content per cell,
where we can compare them to standard 2N but this Is very expensive in terms of
equipment
7. Another suggested method in the case of inter-specific hybrid triploids is the utilization
of isozyme markers that can tell the difference between 3N or 2N due to that banding
patter which we will see, where although it will be the same the concentration of the
information is different

Continue:

x Another very important potential use of triploid which we have not mentioned before is
to restore viability of unviable inter-specific hybrids and has commercial application in
this side where in some cases hybrids if genetic differences are great between them the
viability is not good, but if we induce triploid we might get 100% viability this is due to
use having at least one pair of complete sets of chromosomes such as 2 females and one
male, apparently if we have one complete diploid set from one of the parents it tends to
solve the problem of viability!
x Another potential application of triploids is that the percentage dress out of the fish
looked much better in data than diploids but this is in the situation where we sampled
after sexual maturity has been reached where always triploids have an advantage because
they are not averting energy to sexual development
x Another theoretical idea about 3N we can do them in 2 methods either by breeding them
such as between 4N and 2N or by shock treatments where it has been theorized that those
produced by shock treatment are of less viability and lower quality due to 2 reasons:
o The first being that shock may do damage to the cells which are not reversible and
hence will affect viability of the embryos
o The second being the concept that 3N breed from 4N and 2N are more
heterozygous because producing them by shock which involves retain the
maternal’s polar body will increase the homozogyosty of the 2N individual
produced while in a 4N X 2N we have normal meitotic development and
chromosomes segregation and recombination which is resulting in higher
homosyzgosty and hence better fish
 x In an experiment do prove this done with 2N females and 4N males to produce bred 3N
and 3N produced from heat shock treatments it was always consistent that for both
viability and growth rate, the 4N were the worse, followed by the triploids where both
types showed no differences and the best where the 2N Î hence the theory is not true
x But when the same researcher studied the quality of the eggs of the females they showed
no problem because when mated to normal 2N males they reproduced well, noting that
eggs usually have a tunnel in them called a Micropyle for sperm to enter through it was
theorized that the 4N males produce larger 2N sperm that might not have fit in this
smaller tunnel which was available and truthfully enough a correlation was seen between
fertilization success and the size of the Micropyle where the larger the Micropyle the
more fertile a fish is, so they though that potential ways to overcome this problem is by:
o Either using selection procedures before triploidy to select for females with a
larger micropyle and develop a population from them to use
o Or do a 4N females X 2N male where the bigger egg will defiantly be able to
accommodate the 1N male sperm but this has not yet been done because so maybe
4N female low viability

Section 3
Genetic Sex Reversal:

x What we are trying to do is to produce a mono sex population both phenotypically and
genotypic ally and we are in interested this due to sexual dimorphic characteristics that
we sometimes see in one sex and not that the other which are significant such as higher
growth in male tilapia and in some ornamentals such as beta male fish being much nicer
than females in terms of colors, in some conditions such as salmon, carp and others the
female tends to be a better grower and since in aquaculture we seek to get high growth
this is a good application for it
x The 2nd reason for sex reversal is that it is a way to sterilize fish but we have one sex we
have to sterilize to make its fertility go down to zero in the population in the case of
tilapia even in that case we have a problem with a lot of reproduction in the pond causing
the stocking density to increase hence it depresses growth rates
x In mono sex populations from a conservation stand point if in our environment we don’t
want them to establish we use mono sex the problem is that early in the program we will
require normal brood fish and these tend to risk being let our or loose but the problem is
not found any more after a few generations due to a reduced risk
x If we are trying to use mono sex we always have a risk until we get to single genotype
populations but if we already had a sex reversed (genetically) population and shipped to
us this will remove the risk
x Remembering that we have different sex systems in fish:
o XY system where:
 ƒ Females are XX – homo gametic and if applied to them a hormone they
will become XX males and these will be called sex reversed females
ƒ Males are XY – hetero gametic and if these were feminized using estrogen
they are said to be sex reversed males (so we always call the individual
based on his genes)
o WZ system where:
ƒ Males are ZZ – homo gametic
ƒ Females are WZ – hetero gametic and we can sex reverse them in the
same way above
o Always note that when using hormones we will keep the animal genetically of the
original sex but phenotypically it is changed
x In an XY system when we are trying to produce all females in grass carp where we make
a gynogen XX from the female so we have no an XX population and if not bred they will
die out, if we want to introduce it because of gynogenesis we can maintain the population
by that method by sperms are needed because to activate the egg and usually we tend to
use a sperm of closely related species but not the same so just in case it fails we will not
produce an XY individual and if mistakes occur they will be recognizable of will die,
hence we need to maintain this, closely related species for activation and it tends to be
low yielding but it is overcome by some methods
x If we want to sex reverse to male we use methyl-testosterone where all the fish are
sexually identified by phenotypically we can reverse it by environmental manipulation
via hormonal though feed during the critical sex determination period
x If we want to sex reverse to female we use estrogen and most commonly used is beta-
estradiol and this is also environmentally induced
x Both of these are considered to be fertile but problems do occur with new species, dosage
problems may produce hermaphrodites which are not fertile and since we always have an
exception to the rule but generally it is easier to change female to a functional male and
harder in the opposite direction in both XY & WZ systems
x An example for grass carp of XY system, since gynogenesis produced XX gynogens, we
need to mention here that since at an age of 70-110 mm which is the phase of sexual
determination it is hard to feed the grass carp the treated feed they are implanted with a
hormone releasing implant to sex reverse them into phenotypic males hence now we have
both XX males and females which when mated they will produce 100% female progeny
and each time we took some of the females and sex reverse them to males to perpetuate
the population
o Regarding tilapia the size for sex determination is 11-18 mm noting that size is
more important than age
x Doing gynogenesis each time is efficient but not always possible in all species hence later
on another method is suggested to do this
x Note on anabolic effects Æ if we are interested in this and we look at research depending
on hormone and dosage we might have an anabolic effect were for example if we fed to
male all males with hormone if we are not worried about homo-gametic individuals what
was observed is that XX males and XY males grow both faster than females but a genetic
male will grow faster than a sex reversed females this is why we would be more
interested in producing them not by hormones rather the discussed method
o Also from a conservation stand point we want them to be homo gametic to
prevent sexual reproduction in the wild
x The second method of production of all females of XX is illustrated in the copy book and
involves hormone utilization and progeny testing to identify individual genotypes and
does not use gynogenesis (refer to copy book for this diagram)
x Note: In the environment we have a lot of release of material that mimic hormones in the
environment that are affecting a lot of fish naturally and are causing them to be sex
reversed or hermaphrodites producing ova-testis structures
x We have here a risk of inbreeding due to having a small population we can solve this
problem by bringing in males due to cross breeding and any female works and we can
increase the male produced population to increase the effective population or we can
mate couples to start with to increase the population diversity
x Producing all male populations, to do so we want to produce YY in an XY system noting
that they have been produced and are always fertile and these involve using a sequence of
androgensis, estrogen utilization and progeny testing (refer to copybook diagram) noting
that this is a difficult process to do due to androgensis, noting that female YY in tilapia
are functional but can be different in other cases such as in channel catfish hence we have
other options or 2nd method of all male population production and this involves sex
reversal, progeny testing only
x When we administer hormones, there has been studies to show that the hormone is
cleared from the body after 72 hours from the last administration there is one more
important purpose if we want to do sex reversal by breeding it is because some people
have concern about steroid hormones being used hence it is illegal in some country to
apply methyl testosterone hence the breeding methods are preferred to go around using
them
x There is a time where fish need to be treated to sex reverse them because there is a
temporal effect before which the fish has not got yet activates those genes to determine
the sex naturally hence we should treat appropriately note that some fish and bivalves
tend to have the ability to sex change to maintain a sex ration which is ideal at sexual
maturity naturally in the wild without human interventions
x After the sex determination mechanism has started it is hard for us to sex reverse them at
some point
x In addition it is also important the final size because we need to administer the hormones
for a certain duration and hence the earlier we terminate the treatment the less complete
the reversal we have
x The study on slide 27 shows that the size above 17 mm for the 21 day treatment changed
to 99% male but those under 17 mm where not 100% effective therefore size is more
important that days of treatment hence the treatment should be maintained as the animal
grows from 11-17 mm
x Again regarding the anabolic effect of the sex treatment with hormones where if we have
males those that are males due to hormonal treatment were larger than those normally
males but this is not always consistent hence between species, strains and families the
result tend to vary
x Sex reversal in catfish – if you fee methyl testosterone to the population what happens
surprisingly is that the number of females increase and may reach up to 75% and cause
them to change into females by a process called paradoxical feminization the reason for
this is that they have a bio feedback system which detects the high testosterone and
stimulates the fish to produce aromatase that breaks down the testosterone where the
byproduct of testosterone is estrogen hence having the opposite effect and cause more
females, aromatase inhibitors don’t work very well
x When we look at sex reversal in breeding to produce one sex populations in some species
it works but in some cases it doesn’t work completely because in some fish the sex is
polygenic or a quantitative trait where we have a major loci and a set of modifying loci or
minor that contribute sex determination hence although the major locus may dictate a
certain sex we can have a fish of the opposite sex due to the overriding of the minor loci
and these minor loci are thought to be on the autosomal chromosomes
x Note that the WZ & XY chromosomes are not morphologically different in fish like they
are in humans
x A study showed that in tilapias when we studied individual families where we saw some
families are more female or more male or a 50-50 ratio when we mixed or crossed these
families it showed that a female from a predominantly male family or a female family
reproduced the same sex proportion of higher family, and it showed that a female has 13
rimes more impact on the sex than her male
o Hence this showed that we might also have some of these minor or modifying loci
also on the same sex chromosomes so we might have one female chromosome to
be stronger than another chromosome and hence if one female individual having
on her female chromosome more minor loci with the female alleles than another
then it will produce a larger female population
o These vary greatly between strains as well as families, and it has shown to be a
heritable trait that may allow us to do selection to produce more males or females
 x Temperature influences on sex ratio is also found, where for example in tilapia the
warmer the water the higher the male ratio and what we can do is select fish that are more
susceptible to temperature change to be able to control sex by temperature manipulations

Genetic Engineering:

x What we are trying to do is produce a transgenic individual it is not taking necessarily a

gene from one animal to another but rather than animals that has accepted exogenous
DNA either artificially made or produced from his own DNA and hence a transgenic has
a piece of DNA that it should not be his and we do it for many reasons such as enhancing
production traits, study gene expression, medical applications, transgenic sterilization, act
as a biological factory for pharmaceutical proteins
x The main steps involves:
o Isolate the gene of interest
o Transfer the gene into our organism
o We must obtain expression which needs a good promoter or regulatory sequences
and for it to be properly integrated
o Produce the positive biological effect and then finally to be able to inherit it to
future generations
x We have 2 types that we work with for gene transfer:
o Genomic DNA isolated genes from an exogenous source which is actual DNA
o Or we can transfer cDNA or complementary DNA in this case a major
development is that we can use the reverse transcriptase to produce cDNA from
mRNA since we might not be able to reach the gene and we can pick directly the
DNA that is being expressed since it is using mRNA as a source while genomic
DNA is the full sequences which contains all the introns and exons, but using
reverse transcriptase may be troublesome
x We are basically sometimes need to construct a gene where this produced gene is called a
gene-construct or transgene that we seek to introduce and these are produced by linking
and welding particles to each other and hence may also be called fusion genes and these
include:"
o A promoter needed to regulate transition of 1 gene downstream but now
discovered that some promoters can regulate for more than 1 gene
o Enhancer
o The gene itself containing introns and exons
o Stop sequences
o Linker units
x Note that 1 gene can produce more than one protein due to alternate splicing
x Enhancers tend to enhance gene expression and can determine elevation of gene
expression and can determine elevation of gene expression and can effect multiple genes
 in both directions, introns don’t code while exons do, stop sequences needed to stop
transcriptions
x Promoters can be of 3 basic types:
o Constitutive – these are always on
o Inducible – can be promoting production only when an inducer is present to
trigger it
o Tissue specific – in this case the promoters may only work due to certain tissues
such as only in the muscle or skin so if destroyed using this we can only target
specific tissues
x Some of the inducible promoters tend to be sometimes leaky where they are not totally
inducible where if induced is or is not present it may still express hence leaky
x The whole key behind genetic engineering is to overcome the feedback mechanisms of
the body where for example if we use or duplicate a gene from the same fish into itself
we are basically producing a regulatory system that will not respond to the own body
feedback such as if we increase the growth gene and the 1st expression is increased when
it gets high the body will response to decrease it but the signals the feedback system is
sending will not be received or comprehended due to us having a double gene which
disrupts this signal allowing target growth
x The issue with gene fusion is that we can pleiotropic effect where this fused gene will
effect more than one trait
x By varying bases in promoter we can alter the strength of a promoter, many work was
considered to be done on viral VS fish promoters, noting that viral promoters are very
good and pose no risk since a small piece of DNA will not be infective
x We also have something called reporter genes used to signal if a certain gene integration
has occurred where these genes can be linked to fluorescent markers and show signals if
introduced successfully
x We have some various techniques for gene transfer and they have progressed over time:
o Micro gene injection into eggs where the main problem of this is that it is very
tedious and time consuming, in this case here we are introducing or using millions
of copies of the transgenic that we want to incorporate, usually done at the 2-4
cell stage
o Electro-poration is the next method where the DNA is driven into the egg with
pulses of electricity which are up to 6000 V and they are usually put in trays to do
several in one time and not we can do a Petri dish full of several hundred eggs per
second and hence much more efficient in this method a much small amount of
DNA is used where we need around 50 micro grams of DNA for a few hundred
eggs
o Particle bombardment is the next method where what is used is a gene gun to
blast the cells with the DNA of our interest but I this wrapped in gold (this
method is patented)
 o Tissue injection
o Liposome utilization
o Gama radiation can be used with gynogenesis where this method is considered to
be a shotgun method since although the gamma rays breaks down the DNA into
segments we really don’t know which segment is going to be incorporated
o Sperm mediated transfer, usually was done for mice and people were skeptical if
it works or not but it does where the sperm is coated with the DNA and
introduced via natural fertilization
o Viral vectors, where we can use a virus to introduce a gene, such as a retro-virus
which naturally injects a gene into a cell with the DNA it is not a good technique
at the commercial level because the virus risks becoming active and potentially
pathogenic
ƒ This method is mainly used when the fertilization is internal and we can
manipulate the eggs or sperm
x Another important point is that although we are introducing foreign DNA into a cell each
animal which the insertion takes place in is called a P1 and they are all considered to be
mosaics where this is still not completely understood how gene insertion and
incorporation takes place but we know that apparently it doesn’t occur at the cell 1 stage
since not all the P1 individual’s cells contain the DNA, they tend to have in some tissues
and not others or even within the same tissues some cells may have it while other do not
and this is usually tested by fin clips using southern blot (to be discussed later)
x Since they are mosaics we can’t get a good idea about that genes effect because it is not
in all the cells
x If the animals has the gene in the gonads we produce the F1 generation and it will have
this gene in all the cells and now we can accurately evaluate this animal, IF during
mitosis in the new F1 individual one cell tends to kick out the new inserted gene for some
reason we risk producing a mosaic again but this occurs very rarely
x If the animals that we integrated the gene in didn’t happen in the gonads they will be
worth-less and not used as P1 and we can identify this by gonad biopsy
x Now when we insert the DNA, we can do insertion of both circular and linear DNA and it
has been shown that the linear DNA has a higher rate or chance of success in this process
and when it integrates it tends to coil and circle up forming what is called a concatamer,
this concatemer is in the cytoplasm and is not integrated yet in the Cell’s nuclear DNA or
chromosomes which is what we seek to do, if not integrated these concatamers tend to be
replicated by the cell’s machinery and made into multiple copies and hence there number
tends to increase but as soon as the cell indentifies these very large numbers it will start
destroying them and hence the number is reduced to zero and no integration occurs, in
this situation if a gel is run we will have this segment appearing but it will not be really
integrated and at latter stages of the fingerling we see that these bands will not appear
anymore because they have been completely reduced by the cells machinery +-
x Now if integration took place we can have it as:
o Single copy of the gene
o Multiple copies of the gene
ƒ Æ in addition we can have here that these multiple copies are inserted in
one location or multiple location
x In the case were integration occurred in multiple copies in the same site they are
positioned in a sequences which is called a tandem array which can be:
o Head to tail meaning the different segments are 5’-3’ Æ 5’-3’ and so on
o Or they can be a tail to tail tandem array which is a backward order of 5’-3’ Æ 3’-
5’
x We can also have these multiple copies in different locations hence are said to be
multiple copies in single insertion sites
x Sometimes we risk that the gene we introduce has been broken down and only some
sections of it are inserted and this is what we call partial insertion
x In most studies it has been very clearly shown that there is no correlation between the
number of copies of a certain gene and the level of expression hence if we have 1 or 100
copies of an inserted gene they will have the same effect, noting that exceptions have
been found where correlation has been observed
x Now the inconsistent levels of expression that we see in a transgenic population (example
transgenic salmon showed 10-300% increase in growth variation) due to inconsistent
biological effect is due to potentially may reasons:
o It can be due to the genetic background of the fish to start with
o It can be due to the number of the copies
o It can be due to a the gene insertion being a random process where we cant target
where this gene is inserted so it might have a great effect since we have some
zones which are sequencing and other are non coding and we can have some
zones with enhancers and others are not …
x This also tells us that we need both selection and genetic marker or genetic engineering
techniques to get best results, since now within these trans-genetic population we need to
select those that have superior growth characteristics and use them and cull the bad ones
x With channel catfish and carp we have inserted the rainbow trout growth gene and using
a southern blot we show that integration can occur or nor
x Southern blots Æ use restriction enzymes where we cut the gene with a restriction
enzyme and use a probe to attach to that site to visualize the gene on the gel, if that
sequences was:
o A concatamer (meaning circular looped and not integrated) it will be visualized as
a single band on the gel of a known predicted small size
o If it was integrated it will appear as more than one band of a larger size since
when the restriction enzyme cuts some part of the inserted gene will be attached
 to the nuclear DNA hence the produced segment is much larger and these
segments are called Junction fragments
x To asses different levels of expression we can do it via immune analysis where we see the
differences very clearly within the transgenic population
x The key to success in these programs as we have mentioned before is to be able to
overcome the natural feedback system of the animals where in case of these growth
hormone each cell was capable of producing the hormone to induce growth and was not
based on the hormone produced from the animal’s system
x Using rainbow trout growth hormone gene with trout showed 50-150% increase in
growth, 40% in catfish, 2-4 fold increase in tilapia and usually the utilization of a growth
hormone gene construct is expected to increase growth by 10-90% where various forms
of genes can be used, also to add ion the case of salmon it was seen to increase I by 30%
and some cases showed an increase of 2-30 Fold! Those very high growth rates tend to
decrease survival and increase death
o Î Note that these have not yet been commercially approved
x In salmon the dramatic results is due to the fish showing already very slow growth which
is further depressed by the cold temperatures of the water hence the increase effect of the
growth gene is multiplied where the fish tend to growth much faster earlier which also
tends to give them a magnification effect that they tend to have as an advantage when
they grow, it has also been shown that we might have a pleitropic effect where the
smolting stage of the salmon fish (where naturally the growth rate increases a lot) is
reached much earlier hence it will reach bigger sizes much faster
x It is important to note that due to the evolutionary tree of the animals if we inject a cow
hormone in a fish it will work but now in the opposite method since it has been shown
that when we use hormones in animals done the evolutionary tree they can be applicable
but not the evolutionary tree
x To read Æ chapter on genetic engineering + chapter on combining genetic improvement
programs
x In these examples so far we have only accelerated growth in the initial phase of the
animals and essentially we didn’t get any larger animal at the adult stage (although a few
fish showed a slightly larger size but not very tremendous differences) and hence it was
always shown or said that these methods allow for an increased growth rate to reach
market size faster but will not produce giant fish, BUT in the fish of the Mud Louch
family it was seen that adults were up to 30 times larger than the normal size of the fish at
ADULT stage hence the previous idea is not true anymore but not very common
x Varied results can also be due to as some have discussed due to using domestic VS wild
strains where wild strains since no improvement has been done we have a long way to go
but domestic strains which are already very well improved but this is also not true since
the best transgenic fish in salmon was produced from a cross between wild and domestic
strain which was then gene inserted into it
 x We have previously mentioned that insertion of genes or trans-genetic animals can be
used as bio-factories for some material and these include:
o Scientists were able to produce goldfish luteinizing hormone in rainbow trout
eggs which were harvested and injected into goldfish males producing
spermatization and hence a positive biological effect was shown
o Another case, tilapia was used to produce insulin which was then injected in
diabetic mice and were capable of controlling the diabetic mice sugar levels
o Some transgenic fish also produced clotting hormones similar to those in humans
x Another application of this was in salmon Æ where in the arctic region of the globe due
to the very saline water the water temperature tend to drop below freezing and the water
does not freeze but normal salmon which his farmed when put in such temperature it
tends to die since its blood tends to freeze, but certain fish such as the arctic flounder can
live there because it has a protein in its blood that cats like anti-freeze, genetics took that
gene and inserted it in salmon hoping to be able to produce freeze-resistant fish and
hence push the fish farms up north Æ there have done this and integration was successful
by it has no biological effect since it was not produce in enough levels, but when done
with goldfish it showed better results reflected as higher survival at the lower temperature
range of its temperature range
o Î We should note that these interventions that are done to increase the
geographic distribution of a certain animal by genetic engineering should NOT be
supported since this new animal will be exotic and invasive and may result in a
large genetic pollution in the region that it previously couldn’t live in and hence
reduce biodiversity there.
x The only commercial transgenic fish produced is an ornamental zebra fish where
fluorescent genes from the jelly fish were introduced into it and now it can fluorescent
under light in many colors
x Another application of genetic engineering in fish is to produce disease resistant fish
where by using the Cecropin gene from the moths or pigs (which has an anti bacterial
trait) when inserted in channel catfish these channel catfish when put under certain
disease challenges showed much higher survival than the controls
x Cecropin also had enhanced viral disease resistance in trout and salmon and hence these
methods are seen to be very promising
x Regarding this same topic, lysozymes genes introduce in carp has enhanced disease
resistance and phagocytosis
x Non published data showed that other methods which made us able to Knock-out a gene
dictating for Myostatin in rainbow trout increased growth and in zebra fish when the
dealurase gene (from salmon – responsible for omega 3 fatty acid production) was
introduced it has increased the omega 3 content in the zebra fish

Pleitropy:
x In this section where a transgene which is inserted can affect more than one gene, it is a
analogous to correlation in selection where we can affect that other gene hence when we
have pleitropic effect by proper evaluation we can reduce this risk from affecting other
vital properties
x In growth hormone transgenic (GHT) it is one of the material that tends to affect many
other traits such as fat deposition, osmo-regulation since these are naturally affected by
this hormone
x Insertion of this GH gene tends to enhance feed conversion as well as petite and it was
seen to also effect body shape making it more truncate where usually this is linked to less
dress out percentage but in carp regardless of the enhanced truncate shape we had 1-5%
more dress out and in salmon this was linked to less swimming ability and might be due
to the change from the more swim-able fusiform shape it has but these shape differences
are not easily seen
x In the 30 fold faster growing salmon we saw a pleitropic effect as a condition called
acromalgea which is a problem in the deposition of the cartilage and it also had very big
fat deposits in the guts – hence in the case of salmon we had a negative pleitropic effect
hence these effects can be both positive or negative
x In carp a graph shows that as the percentage increase in weight is show the different
sections of the body tend to change relative to each other hence the whole body
proportion will change since these growths of the different sections although correlated
are not linear to each other
x The insertion of the GHT changes also body composition where it tends to increase
protein deposition and decrease fat hence it becomes leaner, it was also shown that the
individual amino acids will obviously increase in number due to higher protein but the
ratios of the different amino acids tend to change as well and we saw that transgenic fish
had more arginine and methionine and when the different fatty acids where studied the
same conditions appeared but in most cases the differences with most fatty acids where
not statistically different
x Transgenic fish with the GH changed also the muscle internal structure and in salmon and
catfish transgenic fish for the growth hormone showed a higher number of mitochondria
with all the implications of that such as a higher oxygen demand, it also had higher
glycogen and more muscle number cells but they were of the same size
x Sensory evaluation of the transgenic fish showed that there were statistical difference in
taste and texture and these benefited the transgenic fish because it had the superior
characteristics and this could be explained due to the different amino acid ratio that tend
to infer the taste as well as the different muscle alignment structure tended to denote
another texture
x Regarding low oxygen tolerance, when we had GHT salmon it decrease their tolerance to
low DO and this might be due to both physiological and morphological causes such as
the rake number in gills, but these changes in raker number where not consistent in
 different populations hence in some conditions they increased or decreased in number but
they all caused a reduction in the DO tolerance, but in regards to carp the tolerance to low
DO changed based on the method we used to evaluate the animal where if we took:
o Mortality as an indicator – transgenic carp had higher mortality leading us to say
that they are less tolerant to low DO
o If we tested the minutes needed to death at low DO – transgenic fish took longer
time, hence if the experiment was short and only the duration is tested we would
find that transgenic fish had more tolerance
x Gut length in GHT salmon was increased and might be one of the reasons that transgenic
fish grew larger due to the larger surface area to digest and absorb food across
x A pleitropic effect on disease resistance where transgenic GH carp showed parasitic and
bacterial infections to be at a much lower rate but still results are not very consistent
x GHT salmon can also allow us to differentiate between the normal and transgenic fish by
the way they look because one of the pleitropic effects is on their color allowing us to
pick them up directly from a population
x If we look at arrays we can see that GHT tend to affect many other gene expression rates
due to this pleitropic effect

Food & Environmental Safety:

x So far there are not approved transgenic meet being commercially produced for human
consumption although transgenic foods are very common and 70-80% of corn and
soybean produced in the US is transgenic
x 3 main arguments are found against transgenic food consumption:
o Food safety risks
o Potential environmental damage by genetic contamination and production of
exotics
o Unethical (playing GOD) and possibility of risk on animal welfare e (engineering
animals that live at high density)
x For food safety research that has been done has shown that transgenic food in most cases
has no more threat on human safety than normal food, the problem with its consumption
is based on 3 main ideas only one of which is potentially risky:
o Consuming transgenic DNA or protein risk this DNA coming into our system and
eventually integrating with our DNA, although studies have shown that when we
eat something some of this DNA lingers in our circulatory system but it is
eventually left out, and they tend to denature that is why we don’t change to what
we eat
o Some people say that if an inserted gene’s promoter is put next to a toxin
producing gene in fish (such as the puffer fish) it will start producing more toxin,
that is not true because the puffer fish doesn’t have an toxic genes (like all other
 fish) but the toxin produced in it are due to a symbiotic relation it does with
bacteria and algae
o The 3rd and the most important and should be taken care of is the potential
production of material that people may be allergic to due to utilization of gene
constructs from material that man is allergic to hence transferring it to other might
become risky
x One more reason people opposed this is the use of retro-viral promoters and this can be
easily solved since we can simply not use them but use others
x If man was to ingest growth hormones (which he naturally does when he consumes other
animals) he will not have an effect since he doesn’t have the receptors for these hormones
due to him being at a much higher level of evolutionary development and lower material
doesn’t work on him
x The environmental risks, in transgenic plants we had the problem of gametes being
flown by wind and we do have the same problem with fish since they risk swimming
away, and it has always been said that the environmental risk due to a transgenic fish is
related to the fitness of that fish compared to the wild type, the more fit it is the higher the
risk it has on the population, usually the fitness of this animal is established due to:
o Ability to spawn and reproduced
o Ability to evade predation
o Ability to forage feed
o Ability to swim (since it affects all the others)
x Available data shows that different transgenic animals have varied fitness levels based on
the traits we are discussing in addition they tend to differ greatly based on the
environment they are put in where they can be more fit that the wild in some conditions
but not in others
o Example is GHT catfish grew 30-40% large in culture environment and the same
in wild environment this is due to a GXE interaction

GXE Interactions:

x The GXE interaction is when the value or the rank of 2 or more genotypes changes when
they are evaluated in 2 or more different environments, hence the best genotype in one
environment might not be the best in another
x An important practical implication of this is that we are trying to improve fish, hence if
all the work is done in a lab environment and not applied in real life conditions we might
not get the same results, in example of the channel X blue it showed very good result in
the ponds up to 100% better growth but very bad results in small experimental tanks
x These GXE interactions can result in 2 thing or 2 types of results:
o Dramatic changes Æ where the ranking complete flips or is opposite from one
condition to another
 o Not very dramatic Æ where the ranking is maintain in terms of order (absolutely)
but the amounts or differences between them changes, or becomes equal such as
in environment A we had40% difference in performance while in B it was only
5%
x We can predict when we will see a wide GXE interaction effect where the greater the
difference between the compared genotypes or the environments we are comparing the
larger the GXE component, hence when comparing 2 different strains we are not
expected to find a big GXE since genetically very close but when comparing 2N & 3N
the GXE will be wide
x Regarding some performance, GHT salmon showed less fitness when compared in terms
of predation where they were more predated on by controls and their swimming ability
dropped and results for other traits tend to very
x Î Generally transgenic fish are so far shown to be no more fit or even less fit in the
WILD natural environment
x Note on Trojan Effect Æ in Madaca fish it was GHT where since the transgenic males
were larger females were attracted to them more and they had a mating advantage but
their offspring in the wild were weaker hence this advantage to the genetically inferior
population will lead to the extinction of the population, this is a very famous study but
not very accurate since:
o It was produced by a computer model where many other components were not
considered
o Generally in fish the size is not the only factor used to choose the mate because if
it was so then we will always have a selection towards large animals
x The only way for us to reduce risk of environmental contaminations physical
confinement but it always has a risk due to the initially reproductive pair, but the methods
of Transgenic sterilization are making it possible for us to knock out reproductive and
viability genes preventing propagation (but the problem of then not being to propagate it
all is considered) hence we solve it by potentially making these reversible and this will
potentially solve the environmental risk problem.
x The genetic sterilization techniques discussed here are referred to as knock out as oppose
to adding genes which are said to be knock ins, we have many approached to do this for a
method of reproductive confinement and some are:
o Utilization of mutation via gamma rays, where the rays will break the DNA
rending them knocked out but it is a shotgun approach since we don’t know which
will be cut
o Anti-sense approach where we form an antisense gene construct opposite to the
gene we want to know out and insert it and the mRNA produced from it will bind
and render the active mRNA inactive this is not considered ideal since it is hard to
work
 o Ribozymes, here we design a gene that codes for a ribozyme which is an enzyme
that cuts the RNA it is more difficult than other but can be very effective such as
the hammer head ribozymes are thought to be the best
o We can also use cDNA where we insert the DNA of the gene of interest which
will lead to over expression and if to much of it is produced in terms of mRNA it
will disrupt the translation process
o A popular approach now Is the utilization of RNAi which is RNA interferences
this is where we construct a small RNA structure sequences called a hair pin
which contains a lip which bind to the produced mRNA and hence disable the
messenger which is then cut by certain enzymes
o Zinc fingers
o Homologous recombination, where here it is not an anti-sense approach we design
a defective gene and put it in a system called a cassette which depends on
recombination events to splice and insert our gene in place of the one which is
already there, this has a very low rate of probability of occurred and called a
targeted gene knock out, but since fish are so fecund it might occur successfully,
if this occurs in a stem cell culture we can use fluorescent probes associated with
the defective gene to easily identify the mutant cell lines
o Another recombination technique uses something called CRE recombinase to
knock out some genes where we cross a fish with the CRE recombinase with a
transgenic fish and this should produce progeny with the needed gene knock out,
in this process we have 2 fertile line to start with so before mating them we risk
them have escaping and reproducing in the wild and they also risk recombining in
different locations
x So to produce sterilization one study showed success but not mastered yet as a technique
and it tends to vary in results between different lines, the idea is that we have a promoter
then followed by an anti-sense construct of the gene hormone for GnRH which is needed
for gamete maturation, hence it will bind and prevent it from being produce and when we
want to reverse it we inject the actual hormone GnRH to momentarily allow fertility to
occur and they will be fertile only for that spawning cycle, it is labor some and we need
to inject all the fish for all the period before reaching maturation
x The Tet-off system is where we provide sterile constructs where we sterilize the fish by
disturbing embryonic development where escapes may mate but the off springs will die
this is involved in knocking out the BMP2 gene involved in bone development and it is a
promoter in the developmental stage effecting a promoter denoted the name of MAD5
o How it works is that the early promoter occurs in cycle where if teracyline was
added into the system the embryos stayed alive because it interrupted the action of
a certain intermediate in that sequence of reactions , if teracycle was to be added it
will bind with ETA prevent the TA to bind to TRE and acting it and hence
prevent the killing of the embryos
 ƒ The problem with this system is that if we have many gallons of water in
the system we can’t put that much tetracycline which is expansive and
needed to maintain 100 ppm and that AB needs to be processed before
discharged into the system
o These constructs can be of 2 types:
ƒ SF 3 Æ has RNA gene of interest for the other direction
ƒ SF 4 Æ has double stranded DNA in the opposite direction
x Î these both will kill and they can be tested on both the transgenic
and the normal fish since it controls an early embryonic
development gene
x Another approach of this Tet-off is where we try to stop primordial development of germ
cell in the produced embryos hence sterilization occur in the offspring’s and not for the
parents, if tetracycline is added the offspring’s are fertile in the same sequences, this is
much better since we can only treat the brood stock and not all the fish and better for
environmental discharge

Marker assisted selection:

x The whole purpose is to accelerate improvement based on both the market and
phenotypic variation what we know so far if the trait is of high heritability the results
indicate that using marker assisted selection or normal family or mass selection produced
the same result but if we had low heritability marker assisted selection is much better
than the others

Conservation genetics:

x Different agencies involved in conservation have a problem since from one hand they
want to conserve the natural genetic resources and from the other hand they want to
maintain a supply of fish for quality hunting and fishing and these might require us to
change the genetics of certain populations
x Natural population need to be preserved because they have theoretically been selected
for that environment for many years and we see that really aquaculture and natural
conservation go hand in hand because fish farming has allowed us to reduce the pressure
on the fish due to the over-exploitation of the fish stocks that has been indirectly
changing allele frequencies
x And for these genetic programs we need a population to wok with and these resources
for it to use
x But we should note that allele frequencies tend to change naturally in the wild but we
should not stock in a way to accelerate this change and sometime we think that we are
affecting or changing the whole environment why not change the fish to be more adapted
to the environment
 x Some also question, if the wild fish are so well adapted to the environment, then if we
introduce any other fish it should maintain superiority and its allele frequencies should
not be affected
x Usually the higher the genetic variability in a species the higher the ability to adapt and
survive hence it gives them an advantage but in some cases added genetic variability is
not good where we have a small population which is highly adapted to a specific
condition hence if variation is introduced then we might decreases survival
x Naturally selection can take place in 4 different methods:
o Stabilizing – heterozygote’s have an advantage because they generate all possible
genotype combinations and they can stabilize allele frequencies in the system
o Directional – where the population will tend to shift toward one of the
homozygous genotypes and less variation is seen
o Cyclical – where the population goes from one homozygous genotype to the
other in response to environmental stimuli
o Disruptive – where one homozygous genotype is proffered over the
heterozygote’s hence the population will be made o lines of homozygous
individuals
x Sometimes we want to change genetic levels in a population so we want to know what
factors lead to that or to prevent it if in the other situation, usually established
populations are guard to changed but we can adjust size, age, number, frequency and
timing to all effect how effective the restocking is on shifting populations, the best is to
restock completely – some populations in some locations are affected by some factors
more than other (slide 193)

Xeno-gensis:

x GET DEFINITION FROM BOOK ! a process where we use one species to produce
another by allowing gonads of a different species to develop and this occurs in steps:
o Produce 3N host embryos of salmon hence sterile with retarded gonad
development
o Take primordial germ cells from trout male testes into host embryo and they will
migrate into the genital ridge of the 3N salmon and these cells are multi-potent so
they will change to the sex organs of the sex of the host and produce the structure
o Mate the males and females to produce rainbow trout
x Best method to conserve male lines and line in general by only conserving the sperm

___________