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Contributed by British Columbia
Grower's Association:
In this first situation,
we'll deal with the situation where a plant breeder
finds a special individual or clone.
It's a
natural thing to be curious and cross a couple of plants
that catch your fancy. Grow them out and find a new
variation that you like even better. We can preserve the
new variation through cloning indefinately, but
accidents happen and clones die. They can get viruses or
can suffer clonal deprivation from somatic mutations
over time. Plus it's harder to share clones with friends
through the mail than seeds. So it's only natural that
we would want to create seed backups of this special
clone.
But before we start breeding this clone,
we should try and figure what exactly it is we want from
the seeds we are going to create. Do we want them to
simply be able to reproduce individuals like the special
clone? Simple backcrossing (cubing) will accomplish
this. Or do we want to to create seeds that will be able
to create more seeds like the special clone, a true
breeding strain? These are very different in nature. You
see, chances are that your special clone will be
heterozygous for many of traits she phenotypically
expresses. This just means that she will contain genetic
information (genes) for two opposing triats, but you can
only see one, the dominant one. However, her seeds will
only get one or the other of the genes, so her offspring
will express all the genetic information she has,
including what you can't see within herself. If you want
to create a true breeding strain, you need to preserve
all the genes you can see, and remove all the genes that
you cannot, but may show up in the offspring. Creating
homozygosity. The only way to accomplish this is through
selection and generational inbreeding (selecting the
homozygous offspring to be parents for the next
generation).
BackCrossing and Cubing
Backcrossing is where you breed an individual
(your special clone) with it's progeny. Sick in our
world, but plants seem to like it
1) Your first
backcross is just a backcross.
2) Your second
backcross where you take the progeny from the first
backcross and cross back to the SAME parent (grandparent
now) is often called SQUARING by plant breeders.
3) Your third backcross where you take the
progency (squared) from the second backcross and cross
back to the SAME parent (great grandparent now) is often
called CUBING by plant breeders. You can continue the
backcrossing but we just call this backcrossing. Cubing
is in reference to the number three, as in 3 backcrosses
Cubing works on the basis of mathamatical
probabilities with respect to gene frequencies. The more
males you use with each cross, the better the chance
that your reality matches the theory. In theory, with
the first backcross, 75% of your genepool will match the
genepool of the P1 parent being cubed. Squaring
increases this to 87.5% and cubing increases it to
93.75%. You can arrive at these numbers by taking the
average between the two parents making up the cross. For
instance, you start by crossing the P1 mom (100%) with
and unrelated male (0%) getting 100% + 0% divided by 2 =
50%. Therefore, the offspring of this first cross are
loosly thought of as being 50% like the mom. Take these
and do your first backcross and you get 100% (mom) + 50%
divided by 2 = 75%. And this is where we get the 75% for
the first backcross. Same thing applies as you do more
backcrosses. As you will see later, you can apply this
same probability math to specific genes or traits, and
this can have a dramatic effect on your methodology and
selection methods.
Your selection of the right
males for each backcross are the crucial points for
success with this technique. In each case, you could
select males that contain the genes you want, or you
could inadvertedly pick those individuals that carry the
unwanted recessive genes. Or more likely, you could just
pick individuals that are heterozygous for both genes
like the P1 mom being backcrossed. The easiest way to
deal with this is to start by only looking at one gene
and one trait, like lets assume that flavour is
determined by a single gene (in reality it's probably
not). And do some punnet squares to show gene
frequencies through 3 generations of backcrossing. Now
lets assume that we found a special pineapple flavoured
individual in our pine flavoured population that we
wanted to keep. The gene causing the pineapple flavour
could be dominant or recessive and the selection
abilities and cubing outcome will be different in both
cases.
a) pineapple flavour is dominant.
P = pineapple flavour and p = pine flavour
Therefore since each individual will have two
flavour genes paired up, the possible genotypes are PP,
Pp, and pp. Since P is dominant, PP and Pp will express
pineapple flavour while pp will exhibit pine flavour,
these are their phenotypes. Now since the pineapple is a
new flavour, chances are that the special individual
will be heterozygous, or more specifically, Pp.
Therefore, the only possible parent combination is Pp X
pp with the Pp being the parent to be cubed.
Figure 1. The F1 cross
Now most will find it tough to
pick males with the gene for pineapple flavour since
males don't produce female flowers. Therefore, they will
select males randomly and blindly with respect to this
trait. The ratio of P to p genes of the male F1
generation to be used in the first backcross will be
2:6. Another way to look at it is to say that the P gene
fequency is 25%. This means that one out of four pollen
grains will contain the gene for pineapple flavour. Here
is how this plays out in the first backcross.
Figure 2. The B1 cross
Now it's this first backcross that
first creates an individual that is homozygous (PP) for
the pineapple flavour. However, again because of our
limited selection abilities, we choose males randomly.
From the random males we should expect three out of
eight pollen grains to to contain the gene for pineapple
flavour. The P1 female will still contribute one P gene
for every p gene. I'll spare your computor's memory and
and not post the table, feel free to do it yorself
though on paper to be sure you understand what happening
The second backcross (Squaring) will produce
the following:
3 PP 8 Pp 5 pp
Therefore,
68.75% will have pineapple flavour and 31.25% will have
pine flavour. The frequency of the P gene has risen to
7/16 or 43.75%.
And finally, the third backcross
(Cubing) will net the following genotypic ratios:
7PP 16Pp 9pp
Therefore, 71.875% will
have pineapple flavour after cubing has been completed.
Roughly 22% (7/32*100) of the cubed progeny will be true
breeding for the pineapple flavour. The frequency of the
P gene has risen to roughly 47% (30/64).
In
conclusion, if the backcrossing continued indefinately
with random selection of males and with large enough of
a population size, the frequency of the P gene would max
out at 50%. This means that the best that can be
expected from cubing is 25% true breeding for pineapple
flavour and 75% that will display the pineapple flavour.
You would never be rid of the 25% that would maintain
the pine flavour. This model would hold true when trying
to cube any heterozygous trait.
b)
Pineapple flavour is recessive
In this case, P
is for the pine flavour and p is for pineapple flavour.
Convention is that the capital letter signifies
dominance. For the breeder to have noticed the
interesting trait, the mom to be cubed would have to be
homozygous for the pineapple flavour (pp). Depending
where the male came from and whether it was related, it
could be Pp or PP, with PP being more likely. It won't
make much difference which in the outcome.
F1
cross is pretty basic, we'll skip the diagram. We simply
cross the female (pp) with the male (PP) and get
offspring that are all Pp. Since the pine flavour is
recessive, none of the F1 offspring will have pineapple
flavour (hint ). However, the frequency of the gene p
will be 50%.
pp X PP = Pp + Pp + Pp + Pp
Since the F1 generation are all the same (Pp),
the pollen it donates to the first backcross will
contain a p gene for every P gene. The first backcross
will be:
B1 = pp X Pp = Pp + Pp + pp + pp
As you can see, 50% of the offspring will be
pineapple flavoured and the frequency of the p gene is
6/8 or 75%. This B1 generation will generate pollen
containing 6 p genes for every 2 P genes.
Figure 3. The second backcross.
As you can see, the second
backcross or squaring produces pineapple flavour in 75%
of the offspring. And the p gene frequency within those
offspring is roughly 88%. (Remember C88 ). Of the pollen
grains from this squaring, 14 out of 16 will carry the p
gene for pineapple flavouring. When they are backcrossed
to the P1 mom for the third time, they net the following
cubed progeny:
Figure 4. The third backcross
After cubing of a homozygous gene
pair, we end up with roughly 88% of them displaying the
desired trait (pineapple flavour in this case) and also
being true breeding for that same trait. The frequency
of this desired gene will be roughly 94%. If the
backcrossing was to continue indefinately, the gene
frequency would continue to approach 100% but never
entirely get there.
It should be noted that
the above examples assume no selective pressure and
large enough population sizes to ensure random matings.
As the number of males used in each generation
decreases, the greater the selective pressure whether
intended or not. The significance of a breeding
population size and selective pressure is much greater
when the traits to be cubed are heterozygous. And most
importantly, the above examples only take into account
for a single gene pair.
In reality, most of the
traits we select for like potency are influenced by
several traits. Then the math gets more complicated if
you want to figure out the success rate of a cubing
project. Generally speaking, you multiply the
probabilities of achieving each trait against each
other. For example, if your pineapple trait was
influenced by 2 seperate recessive genes, then you would
multiply 87.5% * 87.5% (.875 * .875 *100) and get 76.6%.
This means that 76.6% of the offspring would be
pineapple flavoured. Now lets say the pineapple trait is
influenced by 2 recessive traits and and a heterozygous
dominant one. We would multiply 87.5% by 87.5% by 71.9%
(.875*.875*.719*100) and get 55%. Just by increasing to
three genes, we have decreased the number of cubed
offspring having pineapple flavouring down to 55%.
Therefore, cubing is a good technique where you want to
increase the frequency of a few genes (this is an
important point to remember ), but as the project
increases, the chance of success decreases .... at least
without some level of selective pressure.
Applying the pressure
The
best way to significantly increase your chances of
success is to apply intended selective pressure and
eliminate unintentional selective pressure. Try to find
clearcut and efficient ways to isolate and select for
and against certain traits. Find ways to be sure your
males are passing along the intended traits and remove
all males that do not. This includes ALL traits that may
be selected for. Some traits you will be able to observe
directly in the males. Other traits like flowering
duration you may not. If you are selecting for a trait
you can't directly observe, you want to do some progeny
tests and determine which males pass on the most
desireable genes. I'll explain more on progeny tests
later.
It's important that when chosing your
best males to ignore the superficial traits having
nothing to do with the real traits your looking for. You
see, cannabis has several thousand genes residing on
just 10 chromosome pairs or 20 individual chromosomes.
Therefore each chomosome contains hundred of genes. Each
gene residing on the same chromosome is said to be
linked to each other. Generally speaking, they travel as
a group . If you select for one of them, you are
actually selecting for all of the traits on the
chromosome. There is an exception to this rule refferred
to as breaking linked genes via crossing over, but for
simplicity sake, we will ignore that for now. Getting
back to selection, you could decide to select for a
trait such as you like the spikey look of the leaves
while really being interested in fixing the grapefruit
flavour. But as it may happen, both traits may be on the
same chromosome pair but opposite chromosomes. If so, as
long as you select the plants with spikey leaves, you
will never get the grapefruit flavour you really want.
It's good to keep in mind that each time you select for
a triat, you are selecting against several hundred genes
This is why most serious breeders learn to take small
methodical steps and work on one or two traits at a
time. Especially with inbreeding projects such as
selfing and backcrossing.
Now lets see what kind
of improvements we can make in the first example of
trying to cube a heterozygous dominant trait using some
selective pressure. Lets say that with each generation,
we are able to remove the individuals recessive for the
pine flavour (pp), but can't remove the heterozygous
ones (Pp). If you recall, our P1 mom had the genotype
(Pp) in that model and the F1 cross yielded (Pp + Pp +
pp + pp) as possible offspring combinations. We remove
the two (pp) individuals leaving us with only Pp.
Therefore our first backcross will be:
Pp * Pp =
PP + Pp + Pp + pp
Again we remove the pp
individual leaving us with PP + 2Pp. Going into the
second backcross we have increased our P gene frequency
from 37.5% up to 66.7%. This means that going into the
second backcross 4 of every six pollen grains will carry
the P gene. The outcome is as follows
As you can see, after selecting
against the homozygous recessives for 2 backcrosses, we
have increased our P gene frequency to 58% from 44% in
our squared population. If we again remove the
homozygous recessives, our gene frequency increases to
70% (14/20) going into the third backcross, meaning that
7 out of 10 pollen grains will carry the P gene. Again,
I'll spare your PC's memory and just give your the
results of the third backcross.
B3 cross = 7 PP
+ 10 Pp + 3 pp
This translates to mean that 95%
of the progeny will taste like pineapple after cubing a
heterozygous dominant strain if the homozygous pine
tasting ones are removed prior to to each backcross.
This is an improvent from 72% when no selection
occurred. The frequency of individuals true breeding for
the pineapple flavour rose to 35%. But more importantly,
the P gene frequency improves to 60%. This will be an
important consideration when we discuss progeny testing
.
But for now lets recap the percentage
of individuals true breeding for the pineapple taste in
each of the models. In the case where the pineapple
flavour trait is heterozygous dominant and no selective
pressure is used, cubing produced 22% true breeding
individuals. By selecting against the homozygous pine
recessive, we were able to increase this too 35%. And
finally, when cubing a homozygous recessive gene, we are
able to achieve a cubed population that is 87.5% true
breeding for the pineapple flavour. And as I pointed out
earlier, these numbers only apply to single gene traits.
Lets say the pineapple flavour is coded by two seperate
genes, one dominant and one recessive, and you are able
to select against the homozygous recessive pine flavour
while selecting for the dominant pineapple flavour gene.
Your cubed population would then contain 87.5% * 35%
(.875 * .35 * 100) = 30% true breeding individuals. As
you can see, as long as the cubed source is
heterozygous, it doesn't matter how many backcrosses you
do, you will never achieve a true breeding
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