I'm going to explain how geneticists
use genetic techniques to identify
alleles (loci) that might contain
incompatible alleles. This concept is
sometimes known as transmission ratio
distortion and segregation distortion
Understanding how this analytical
process works requires us to review a
little bit about meiosis and the
generation nomenclature in genetics. So
the way that this diagram is organized
and I'm going to be filling this in as
we go through this demonstration is: I've
got generations listed on the left, with
the P0 or parental generation shown at
the top, and then I'll diagram
individuals from an F1, the offspring, and
then the F2 generation, the
grandchildren, and the somatic genotypes, 
the soma are cells in the body that are
any cells other than the gametes, which
includes sperm and eggs, and I'm not
indicating here which type of gamete is
a sperm or an egg -  it doesn't really
matter for this example.
So what I'll diagram here are genotypes,
and I'll diagram cartoons of chromosomes
from two different individuals of the
same species: parent A and parent B, and
inside each nucleus (or cell) of each
somatic cell example and each gamete,
I'll draw cartoons of two different
chromosomes, which I'll call chromosome 1
and chromosome 2. So here in the P0
generation I'm assuming that parent A
has particular genotypes of chromosomes
that are diagrammed as being red
chromosomes, and in this case red stands
for a particular DNA sequence that's
different from the chromosome sequences
found in the other individual of the same
species: parent B. So while these are
meant to be two individuals, A and B from
the same species, the different colors of
their chromosomes indicates that those
chromosomes can be distinguished based
on the DNA sequence of those chromosomes. In other words, the parents are
genetically different. And so I'm going to
use the colors of the chromosomes to
track what happens when these
chromosomes undergo meiosis (the first
step, the arrow heads) and produce the
gametes from individual A and the
gametes from individual B, and then we'll
see what happens to those chromosomes
when those two gametes fuse at
fertilization to produce F1 offspring.
and before I go any further I should
mention that it's an extreme assumption,
which is almost never going to be true,
that any parents would be homozygous -
that is both copies of the same
chromosome, shown as the two horizontal
lines (two copies of chromosome one, the
shorter chromosome, and, in this case, two
copies of chromosome 2, the longer) would be genetically
identical, which I've diagrammed here as
both of them being the same color - both
being red. That's very very rare, probably
impossible in most species, but we use it
here as an assumption to make the
diagramming easier. So here what we can
see is, in meiosis, what happens is: the
gametes have half as many chromosomes.
They have one of each instead of two of
each, as in a typical somatic cell. So in
the process of meiosis, one of the two
copies of a chromosome will be put into
the egg or sperm (the gamete) and that's
true of every single chromosome. So in
this case, that's how those products of
meiosis (the gametes) are produced. And so
because individual A only has completely
red chromosomes, there's no way that any
gamete other than a totally red gamete
(sperm or egg) can be produced, and the
same is true of parent B. So if we know
that parent A is, as I mentioned earlier,
totally homozygous (all of their
chromosomes are identical: chromosome one,
the two copies of the chromosome are
exactly the same; the two copies of
chromosome two are exactly the same)
then the geneticist can predict (we know)
that there's only one possible type of
gamete. It's going to have the same genetic
makeup as the parent somatic cell. And
that's why I know here that the gametes
that I've drawn in the P0 generation
are all of the possible haplotypes,
haplotype being a fancy word for
"genotype of a haploid cell" or gamete. So
in this case we know that's all of the
gametes that parent A will make are
gonna be like this: red chromosome 1,
red chromosome 2, and parent B can only
make blue chromosome 1, blue chromosome 2 type gametes.
Likewise, you should be able to predict
what's going to happen when these two
gametes fuse to re-produce a diploid cell
through fertilization.
So what is going to be drawn in the
somatic cells of the F1 offspring when
these two gametes fuse at fertilization?
The offspring inherits a red copy and a
blue copy of chromosome 1 and a red
copy and a blue copy of chromosome 2. So
in this case, in this specific type of
cross, each F1 offspring (here I've shown
two different individuals) they're
genetically identical, and that's because
all of the gametes of the P0 parents, in
this case, are identical. So every single
fertilization event between parent A and
parent B will involve red chromosome 1
and red chromosome 2 and blue chromosome
1 and blue chromosome 2. So all of their
F1 offspring will be genetically
identical to each other. And here is one
of the first critical concepts of
understanding how to use genetic crosses
to look for genetic incompatibilities. In
the F1 generation, if we turn our
attention to the graph on the right, what
a geneticist would do to understand what
the expectation is for alleles (the types
of chromosomes that are present in
offspring, which is what we're going to
eventually do experimentally in the F2
generation)… here in the F1 generation we
can look at each spot on each chromosome, so we could look at, for example, the left
end of all of the chromosome 1s that
exist in this generation, in this case
two individuals, and we could plot at
that locus on chromosome 1, the first
little segment, what percentage of those
four chromosomes are red. So we have 1, 2,
3, 4 chromosomes (4 copies of chromosome 1 in this generation): half of them are red.
So we've got a and b, for example, little
a and little b - just using a different
method of counting chromosomes - so we've got 2
copies of a red block of chromosome and
c and d: 2 two blue. So 50% of the four
chromosomes are red so we can plot that
in the graph on the right. The top would
be 100% red, the bottom is 0% red, so
we're at 50% red: that first little
segment of chromosome 1 is 50% red. And
then we can move to the next block and
it's still 50% red, and we move that way
across the small chromosome, chromosome 1,
and every time we look when we count up
the red and blue across all the copies
of the chromosomes in that generation, we
find that there's a hundred percent (100%
of the time, rather) it's 50 percent red,
50 percent blue. Again, this shouldn't be
a surprise, because we know that the
contributions of genetic material from
the gametes into this generation were 50
percent red and fifty percent blue, so
this first step is pretty trivial,
but it's important to note because it
sets our expectation. And evolutionary
geneticists use this as the null
hypothesis, or expectation, that if you
start with a hybrid individual or a
series - a population - of hybrid
individuals that have a 50% red genotype,
then if nothing evolutionarily happens (is selected for or against, in terms of
the genotypes of these individuals) then
as time passes, in an ideal situation
(like no genetic drift), in the F2
generation we should still see of all
the individuals in that population in
the F2 generation, however many there are,
we should expect to see the same
frequencies: on average, half of the
chromosomes should be red, and half blue.
And that's why down here, in these graphs
that I've created, when we look at the f2
generation there are these gray lines.
Those are these expectations that we
came up with from the F1 generation: that
all else being equal, if no genetics are
being selected for or
against (no genotypes), then the allele
frequencies (the percent red at every
part of every chromosome) should stay the
same:
50% red, 50% blue. In other words, if we
were to see a bias, or some sort of plot
that showed there were deviations from
that expectation, those deviations
"segregation distortion" or "transmission
ratio distortion" is suggestive that
there could be selection acting on
certain genotypes. This would mean, for
example, if we saw peak there, where a
hundred percent of the organisms in a
population in one generation only had
red alleles, that might tell us that it's
beneficial for those organisms to have
red alleles, or similarly it could tell
us that it's bad to have the blue
version of that chromosome and that's
why there are no individuals left in the
population (or existing in the population)
that have the blue allele. So in summary,
this is the concept of transmission
ratio distortion. We generate an
expectation, and then we experimentally
look at hybrid genotypes: we genotype a
bunch of individuals, and then we plot
the distribution of genotypes across one
or multiple chromosomes and we look for
regions where there's statistically
significant divergence from the observed
values and the expected values. So now
it's time to think about the second
round of meiosis and what happens when
the F2 generation is produced. And before
we get there, there's one other concept
to know about (or to remember), and that's
the fact that when meiosis occurs,
chromosomes can recombine with each
other. And, simply, what that means is
during the process of meiosis,
chromosomes can break, which I'll diagram
with a little X and exchange arms
(pieces of chromosomes). So, in this example,
during meiosis what would happen is: that
pure red chromosome
in the F1 individual would break into
two parts, so the left half of the
chromosome and the right half of the
chromosome, and they would physically
recombine, or stick together, with the
equivalent pieces of the blue chromosome that were created when both copies of the
chromosome broke. So this is something we didn't see in the F…in the, sorry, in the
P0 meiosis creating the F1, because
recombination still happens up here, but
we can't tell, because what does a blue
chromosome look like when it
recombines with another blue chromosome? You produce yet another all blue
chromosome. So meiosis was seeing
recombination, but we can't detect that
genetically, when a parent is homozygous -
when their two chromosomes are
identical. Now in the F1 generation, we
have a heterozygote: an individual whose
two copies of the same chromosome,
chromosome 2 for example, are different.
We can detect when recombination happens
by seeing chromosomes that have some of
the red DNA sequence and then the rest
of the chromosome carries on with the
blue version of the chromosome sequence.
So now we can see how I've diagrammed
possible (really important) example
recombinant haplotypes. Recombination is
a very complicated process that can
create a lot of different genetically
diverse chromosomes. In this case, we
could imagine that recombination in
chromosome 1 happened there,
on chromosome 2 happened around there,
chromosome 3, chromosome 4, and I'm
leaving out a lot of subtle details
about how recombination happens and
about how meiosis works, so for more
information check out videos or other
resources that explain those concepts in
greater detail. But the point here is that
we have example gametes of the F1
individuals, so one F1 sperm for example,
let me be a little bit more explicit, let's say
that's a flagellated sperm, and let's say
that's an egg (singular - an ovum),
then the sperm has a recombinant copy of
chromosome 1, and a recombinant copy of
chromosome 2, and the same is true of the
two versions of the chromosomes that are
in the ovum from parent B. So now we're
talking about the F1 individual, so let's
instead refer to them as, say, individual
C and individual D. So individual C
produced many, many sperm certainly in
their lifetime, but here's just one
example - the possible haplotype (genotype)
of the sperm, and again, individual D, a
female, produces many Ova in her lifetime,
but this is one example. And like before,
we know, based on how I've drawn these
gametes, what the F2 - the offspring of
C and D, the F2 child - grandchild of the
P0 generation as well - will look like.
It'll be the two copies of chromosome
one, and the two copies of chromosome two.
So right now we're at the point where
we're starting to look at F2 genotypes
and remember: the idea here is to look at
an F1 to generate the predicted (or
expected) frequency of chromosome
fragments of alleles: red versus blue, and
what we're going to want to do is to
look not just at one F2 individual but
lots of F2 individuals and then plot the
average percent red across, say,
chromosome 1 and chromosome 2 and look
for deviations from this gray 50 percent
expected line. So before we get to
multiple F2 individuals, let's just check
out this one individual right now and
see if they have a genotype that holds
pretty much to expectation. So again, let's
do one or two genotypes really quickly
as a demonstration. So if we look just at
chromosome 1, you start on the left
side. Because there's only one individual
in the population at this point in time,
there are two copies of this chromosome
1: copy one, copy two.
So, half of them are red, that's 50% red, so
that matches our expectation, so
that doesn't make it look, yet, although
this is just one individual, doesn't make
it look yet like there's any
transmission ratio frequency distortion,
or segregation distortion. if we look at
the right side of that chromosome, one
red and one blue, that's 50% red, and
then we could plot everything in between
those two. On chromosome 2, the left end
is 50%, right end is half red (one copy's
red, one copy's blue, so 50%
red) 50% red. And so on. So we do the same
plot for all the spots in between, and we get
a plot that looks like this. So why does
it look like this? Well, part of the
reason that there are these deviations,
like an extremely low value - it looks
like almost all blue, and an extremely
high value (entirely red or almost
entirely red) on the second chromosome is:
that there is a spot on the first
chromosome that is homozygous: both
copies are blue. So that's that spot, and
likewise, there's a region of chromosome 2 that's homozygous in this F2
individual: 100% red, and that
corresponds to that region. And this is
why it's important to look at multiple
individuals, because any one individual
will have lots of regions of their
chromosomes that are homozygous blue or
homozygous red, and that will make these
plots sort of extreme-looking. The only
possibilities are 0%, 50%, or 100%
if you look at a diploid
organism that only has two copies of one
chromosome. But if we just stop here and
think about how we would interpret this
data (these data) if we just only had one
individual to look at: this could look
like there is, well there is 
segregation distortion, with an "n" of one
(just one example individual), but it would
look like this individual has a blue
genotype in the middle of chromosome 1
and a red genotype in the middle of
chromosome 2.
So what that could suggest in an extreme
example is that all of the individuals
that happen to get red homozygous
genotypes at that spot on chromosome 1,
and all of the individuals that had blue
genotypes in that region of chromosome 2,
didn't exist. Maybe they died, maybe the
embryos were never developed…something
happened to remove all the individuals
with that particular combination of
genotypes from the population. In other
words, it's individuals that would have
made this plot balance out - individuals
that were red where there's a trough
there, and individuals with the blue
genotype (0% red) in the middle of
chromosome 2, opposite where there's a
peak. So that is an indication of: that's
a possible set of incompatible alleles -
that combination of genes (or alleles) was
never seen in this one individual and, of
course, again, this is why we have to look
at multiple individuals, because one
individual just doesn't provide enough
data to get a good sense of whether or
not this is a statistically significant
bias from the expected value of 50%. So
here, now, we have 8 individuals. I've
added, as it says here, 7 more random
genotypes, right? These are random
genotypes, because, remember: the gametes
that were shown (the F1 gametes) are
potential recombinant haplotypes. These
are not the only possible combinations
of recombination breakpoints and
versions of chromosomes 1 & 2 that could
be put into gametes by the F1
individuals that produce them. And so
these, by extension, are definitely not
the only possible genotypes of F2
individuals: it's just a random set of 8
that I came up with for this example. And
now, yet again, we can do the exact same
analysis. We could start at, say, the left
end of chromosome 1 and make our plot. So
we have how many…let's see…we have 10 in…or sorry… eight individuals,
so we have two times eight or sixteen
different chromosomes. So let's count how many of those sixteen are red at the
left end of chromosome 1. 1…2…3…4…5…6…7…8…
and 9: so 9 out of 16, or a little bit
over 50%, red. I'm pretty close to the
expectation, and if you keep going
through all the chromosomes for all
these individuals, this is the plot that
you'll obtain: no significant deviation
from 50 percent. The line wanders a bit
around 50 percent, but there's nothing
extreme that warrants additional
attention, that would suggest that
there's maybe a pair of alleles that are
incompatible with each other. So, this is
an example of what it would look like,
under normal circumstances (where there's
no evolutionary selection on any
particular genotypes or combination of
genotypes). So now I'm going to switch and
show you an example of what happens when
there is selection on a particular
combination of loci.
So, for the purposes of this example,
let's say that it's really really bad
for your health if you happen to be a
hybrid that has the blue alleles on the
right end of chromosome 1 and the red
alleles on the left end of chromosome 2.
So blue on 1: say there's a gene there,
gene X, and if you have the blue version
of gene X and the red version of gene Y,
say, that destroys your ability to produce
cellular energy and so at fertilization
this cell will never even divide into a
second cell, much less become an embryo
or develop into a living adult organism
that can reproduce on its own. So in this
example, if that was the case, if every
individual that has blue on the right
end of chromosome 1
and red on the left end of chromosome 2
dies before we have a chance to genotype
them and measure their genotypes, then all
three of these individuals from that F2
generation we won't see. We won't be able
to collect DNA from them in the
laboratory if, for example, if they're
embryonic lethal. We'll never get an
adult organism to collect DNA from to
measure, and what happens here is: this
has an effect on the graph we're going
to produce, because now we're not doing
the graph shown in the middle, where we
were looking at the genotypes of eight
individuals. Now we've got five
individuals that are representative of
the population, but there's going to be
some bias that we can see in the plot,
because we've gotten rid of all of the
individuals that are homozygous blue on
the left end of chromosome 1 and also
homozygous red on the left end of
chromosome 2. So let's check out what
this plot looked like. So, when we
experimentally determine the genotypes
of all of the living organisms (5 in this
F2 generation) in this case - when we
genotype all of their chromosomes (we
might do this, for example, by using PCR
or deep sequencing technology to figure
out the genotypes of each chromosome -
each pair of each chromosome - in each
individual) we see that there's an
under-representation of red at the left
side of both chromosome 1 and chromosome
2, and if we look at the genotypes we can
see that's true. Individual one up here
at the top is heterozygous: they're red
and blue, so there's a little bit of red
at the left ends of chromosomes, but most
of the other remaining individuals are
homozygous blue on the left end of
chromosome 1. Here's another
heterozygote. So there's a little bit of
red left in the population, but because
individuals that were (and because of
recombination, which is tricky thing in
this case) individuals that were blue/blue
on the right sides of the
chromosomes were eliminated. That allele
combination is most often associated with
being red/red on the other end of the
chromosomes: a blue-blue, recombination
breakpoint, red. Blue, recombination
breakpoint, red. So this form of selection,
which is natural selection - individuals
that have that particular combination of
genotypes on two different chromosomes
(chromosome 1 and 2) then exhibit a
bias in the transmission of alleles, the
transmission of red and blue from the F1
generation into the F2 generation, or at
least the F2 individuals that are alive
and that we can study. So this is the
essence of using genotyping of hybrids
to try to identify regions of
chromosomes that contain genes that
might be involved in genetic
incompatibilities: genetic interactions
between red and blue, for example, that
would kill particular genotypes of
organisms, or make them sick. So finally,
if we turn our attention back to the
allele frequency plot in the lower right,
there are a couple of different
interpretations of what these data mean.
There is bias at four different loci,
and we would have to do statistics to
understand if this was statistically
significant bias, and in this case I'm
assuming that's the case. And so there
are four loci: the left and the right
ends of both of the chromosomes look
like they're significantly skewed (or
deviated): this is the segregation
distortion or marker transmission ratio
distortion, depending on what you want to
call it. And this is compatible with two
different interpretations. Based on the
plots that we see. And we've already
talked about this briefly, but here's the
idea: I've told you that my scenario was
that blue on the right end of chromosome
1 and red on the left end of
chromosome 2 was the incompatible
combination, and that's that pair of
alleles. So what you would expect to see,
if transmission ratio distortion is
causing an incompatibility - and this is by no means proof of that,
more experiments would need to be
performed to prove that it's marker
transmission ratio distortion
that's causing a genetic
incompatibility, but one trend is shown
here:
that's the homozygous blue and red is
bad, so we see only red genotypes (high
peak there on chromosome 2) and
only blue genotypes (or mostly blue
genotypes) on the left end of chromosome 2.
And at the same time, we see that there's
another high-low pattern where there's a
peak on one chromosome in a valley, or a
trough, deviation from the expected
frequency of alleles on chromosome 1,
and that is because of the individuals
that were eliminated. Not only were they,
as already drawn, blue on the right end
of chromosome 1 and red on the left
end of chromosome 2; they also happen
to be, as you can see, these eliminations
were also of individuals that were red
the left end of chromosome 1 and also
blue on the right end of chromosome 2.
And that is why that valley and that
peak are present in this plot as well. So
if we have actually observed these data
and we saw a plot like this, we would
have a lot more work to do to try to
interpret which, if any, of these
combinations of genes is epistatically interacting. That is: causing
some sort of hybrid dysfunction that
might be killing particular genotypes of
hybrids. So this has been an introduction
to the concepts and basic practices of
detecting genomic regions that can
potentially play roles in hybrid
incompatibilities or negative epistatic
interactions by producing allele
frequency plots, looking for transmission
ratio distortion, and then segregation
distortion. Thanks for watching.
