Gene Linkage and

Recombination
Do chromosomes have anything to do with genes and inheritance?
Yes, of course we know that now, but at one time it was not certain.
Experiment with corn…
C colored seeds c colorless seeds
Wx makes amylose and amylopectin wx makes only amylopectin
In one strain chromosome 9
had part of chromosome 8
attached to it, and it had a
knob on the other end. This
structure could be observed
under the microscope. It
turned out that in this strain
the chr 8 translocation and
the knob were cytological
markers for genes nearby,
namely C and wx.
Whenever a plant got the
knob, it was C. Whenever it
got the chr 8 translocation,
it was wx. Genetic markers
were moving with
Conclusions:
cytological marker.
Moreover, sometimes new 1. Genes that caused specific phenotypes were
chromosomes were associated with chromosome transmission.
observed (recombinants),
2. Chromosome structure could sometimes be
and the phenotype was
reorganized (recombined) to generate new
predictable.
forms.
Human Chromosomes
Metaphase stage of
Mitosis
Chromosomes are
highly condensed and
can be observed
microscopically.
Stained to generate
unique banding
patterns.
Organize by size and
banding patterns
Karyotype
Fluorescence In situ hybridization (FISH) and “Chromosome Painting”
Linkage of Genes

If every gene sequence was its own little chromosome, there

would be about 25,000 little chromosomes. Each of the 25,000
little chromosomes would come in a maternal form and a
paternal form, and would segregate to either side of the cell
according to how they happened to be lined up. Things could in
principle work this way, but they don’t.
In reality there are only 10,20,30 or so chromosomes, which
means that there are hundreds to thousands of gene on each
chromosome.
These genes are ‘linked’ to one another through stretches of
intergenic nucleotide sequence. The chromosomes you get from
mom contain your maternal alleles, and chromosomes you get
from dad contain your paternal alleles.
Imagine this….

Mom’s Mom Genes Meiosis About Half Mom’s Mom,

Mom’s Dad Genes Half Mom’s Dad
Diploid Haploid
Everybody segregates independently

Or this….
Mom’s Mom

Meiosis OR
Mom’s Dad

Here Genes are Linked and you get

all Mom’s Mom or all Mom’s Dad
Mom’s Mom Some of Mom’s Mom, some of Mom’s Dad

Meiotic OR
Mom’s Dad Recombination

Here Genes are Linked but are Mixed and Matched

by Meiotic Recombination
You get some of Mom’s Mom and some of Mom’s Dad

The concept of linkage means that when you get a grandma allele, the
surrounding gene alleles are likely to also be grandma’s. If you get a
grandpa allele at some locus, the surrounding genes are likely to be
grandpa’s. Of course meiotic recombination mixes these up, but such
recombination only occurs a few times along the length of a
chromosome. This means the chance of two closely linked alleles on an
original chromosome getting separated and not transmitting together is
pretty low. This provides a basis for mapping disease genes, and
mapping gene order.
 Maternal Maternal Paternal Paternal
Grandma Grandpa Grandma Grandpa
gives this gives this gives this gives this

Maternal Alleles

Paternal Alleles

Maternal Alleles

Paternal Alleles
PARENTAL MIXER

Diploid Mom Diploid Dad

Grandma Alleles Linked Grandpa Alleles Linked

Grandpa Allele s Linked

Grandma Alleles Linked Grandma Alleles Linked

Grandpa Allele s Linked

Grandpa Alleles Linked
Grandma Alleles Linked
Grandma Alleles Linked

Maternal Paternal
Chromosome Chromosome

Person composed of mixture of grandparent DNA

Distribution of Genes During Meiosis

Case 1: One gene, 2 alleles S and s

Gametes will be S or they will be s. Fine when talking about single gene
Case 2: Two genes, two alleles, genes sort independently (on different
chromosomes) S and s (e.g. chr 1) and T and t (e.g. chr 2)
S and T S and t s and T s and t
This is the Mendelian situation
Case 3: Two alleles, genes are linked together on the same chromosome
Allele S linked to allele T on one chromosomal copy
Allele s linked to allele t on the other chromosomal copy
If organism is S it is also T (ST)
If organism is s it is also t (st)
Case 4: Recombination would generate nonparental genotypes
(recombinants) St and sT
Example of genetic linkage
is illustrated using X-linked
genes in Drosophila
2 genes, each with 2 alleles:

XW red eyes
Xw white eyes
XM normal wings
Xm minature wings

Genes are LINKED along the

X chromosome
Question is how the eye
color and wing phenotype
genes will be transmitted
due to the fact they are
linked
Suppose you started with a female fly that was a heterozygote, XWM Xwm
If linkage meant nothing, we could consider XWM to be two independent parts,
XW and XM. Likewise, we could consider Xwm to be two independent parts, Xw
and Xm. In this case XW could segregate with either XM or Xm, and Xw could
segregate with XM or Xm. In such a case the alleles at two linked loci would
act in a Mendelian fashion. The types of offspring would be 50% parental
(XWM or Xwm) and 50% recombinant (XWm and XwM).

Is this what happens?

No.
Suppose we again start with a female fly that was a heterozygote, XWM Xwm
If linkage was absolute, then W and M would always be transmitted together
and w and m would always be transmitted together. In such a case the
offspring would be 100% parental (either XWM or Xwm) and 0% recombinant
(XWm and XwM).

Is this what happens?

No.
Suppose we again start with a female fly that was a heterozygote, XWM Xwm
If linked genes sometimes transmitted together but were sometimes unlinked
or separated by meiotic recombination, then W and M would usually transmit
together, and w and m would usually transmit together. But sometimes we
split them to generate new chromosomes with genotype XWm and XwM. In
such a case the offspring would be some % parental (either XWM or Xwm) and
some % recombinant (XWm and XwM).

Is this what happens?

Yes!
Here we set the experiment up…

And why do we always show parents and F1s like this? Because in
actual experiments you need to begin with true breeding parentals you
have stored in the lab in order to make the heterozygous parents you
want to use in your real experiment.
 W w W W w w
replication
This figure shows
the idea of what M m M M m m
happens during
meiosis for genes Xpat Xmat
that are linked.

W w W w
crossing
over
M M m m (chiasma)

synaptonemal complex

OUTPUT

W w W w

M M m m

recombinant
chromosomes
I know this is too
small to see, but it
shows all sorts of
possible
recombination
events that could
occur during
meiosis, along with
the number of
recombinant and
parental
chromosomal
configurations.
 What is the chance that recombination occurs
between two linked genes?

That depends on how far apart the genes are, that is, how
much sequence space there is for a recombination event
to occur.

Here, four chromosomes have been aligned. The greater

distance between B and C means there is more chance for
crossing over between them compared to B and A, which are
closer together.
By definition, 1% crossing over, as measured by the number of
recombinants, is 1 map unit (1 centimorgan)
The total number of recombinants cannot be greater than 50% of
the total number of progeny. 50% is the value one would obtain if
the genes are unlinked (on separate chromosomes) or are so far
apart on one chromosome that they appear to be unlinked. That is
to say, with 50% parental and 50% recombinant you cannot tell if
the genes are on separate chromosomes and so they sort
independently, or if they are linked but are so far apart that meiotic
recombination between them is so frequent that they appear
unlinked.
Here, C and D seem unlinked, but can be connected by
another gene E that is linked to both.

45%
25%

C E D

70 cM
Keep in mind that in meiotic recombination we suppose that ANY of
the two chromosomes on one ‘side’ (as drawn in two dimensions on
a piece of paper) can recombine with either of the two chromosomes
on the other ‘side’. This depiction may not accurately reflect the 3-
dimensional configuration of the chromosomes in the actual
synaptonemal complex.

X X
X X X X
X
X X XX
 A two point testcross to look at recombination
between linked genes

a+b+ / a+b+ X ab / ab

Gives heterozygotes a+b+ / ab

In a test cross we would cross these heterozygotes against
homozygous recessive
So, a+b+ / ab X ab/ab Result shown on next slide
Why do we testcross against homozygous recessive? Because if the
parent being tested transmits a wild type +/dominant allele, we can
observe that in offspring as dominant. If the parent being tested
transmits a recessive a allele, we see that in offspring as the recessive
phenotype. To understand this turn it around a bit and imagine that
instead of homozygous recessive, test crosses involved homozygous
dominant. What happens?
Start with true breeding
homozygous dominant and
homozygous recessive.

Obtain heterozygous F1

Testcross the F1 to produce

the F2:
Some chromosomes are the
same as the original
parentals
Some chromosomes have
undergone recombination
between the a and b genes
to form recombinants
 Mapping Gene Orders and Distances Using a
Three Point Test Cross

A standard mapping problem of this sort is where you consider three

genes, a, b, and c and you wish to use a testcross (against homozygous
recessive at all three loci) to determine:

1. what is the order of the genes along the chromosome?

abc, bca, cab
Note that these are NOT the same

2. how far apart are the genes from one another?

 A Simple Three Point Test Cross Problem
Three genes…..linked? order? distance?

+++/abc X abc/abc

Notes:
1. The point is to determine gene order and distance.

2. In this example you are given the fact that one chromosome is all wt
(+++) while the other chromosome is all mutant (abc).

3. A testcross is by definition against a test organism that is

homozygous recessive at all three loci. As noted before, the purpose of
this is so that you can see the phenotypic output of meiotic
recombination events.

4. We don’t just magically ‘get’ the +++/abc organism. We have to

prepare these from +++/+++ and abc/abc stocks beforehand.

5. The fact that the problem is WRITTEN abc is irrelevant to the actual
gene order. It could be abc, bca, cab, but you do have to write it down
somehow to begin with.
 Results
abc 179 parental types (book uses letters ‘p’, ‘j’, ‘r’)
+++ 173 parental types

+bc 46 recombinants due to single crossover

a++ 52 recombinants due to single crossover
+b+ 22 recombinants due to single crossover
a+c 22 recombinants due to single crossover

++c 4 lowest frequency recombinants—double crossover

ab+ 2 lowest frequency recombinants—double crossover

Note that the pair of recombinants with the fewest numbers are
assigned as resulting from double crossovers.
Also note that the pair of progeny with the most numbers represent
the parental alleles (in this case all the +++ are together on one
chromosome and all the recessive alleles abc are present on the
other chromosome)
One way to work this problem is to ask which of the three possible
orders would generate ++c and ab+ as a result of double crossover.
Each of the following is a possible order: abc, bca, cab. For each
possible order we show what the genotypes of the double recombinants
would be. Then we match these possibilities with what the double
recombinant genotypes actually are. Whichever order generates the
correct set of double recombinants is the correct gene order.

X X X X X X

The order (bca) generates the correct double recombinants.

Here is what happened to generate parentals, single
recombinants, and double recombinants.

no X over 173

179

single 22
X 22

single 52
X 46

4
double
X X 2

500 TOTAL
The last step is to see how far apart the genes are. Since the
frequency of recombination is a measure of sequence distance, we
can use that.

Recombination frequency between b and c

(22 + 22 + 4 + 2)/500 = 0.1 (10%, or 10 map units)

Recombination frequency between c and a

(52 + 46 + 4 + 2)/500 = 0.208 (21%, or 21 map units)

Recombination frequency between b and a

21 + 10 = 31% (31 map units)

Afterwards you draw out the map.

Notes:
The number of double crossovers should be the product of
single crossover frequencies (0.208 X 0.10 = 0.0208) 2.08%

However, the observed frequency is only (4 + 2)/500 = 1.2%

The reason is that having a single crossover reduces the chance

that there will be an additional adjacent crossover.

Counting the recombinant progeny actually underestimates the

number of crossovers and thus the map distance. That is, some
crossovers, such double crossovers, occur between two genes
and end up just to restore the parental configuration.

Another Note: In these problems you need not expect all the
+++ to be ‘on top’ or ‘on bottom’. Instead, the starting parental
situation could be a+c/+b+, etc., and you would need to figure
that out.
Suppose you know a disorder is genetic (inherited).
How do you find the gene that causes this disorder?
You have say 25,000 choices. Which one? The
genome is huge. This is a difficult problem.

Genes can not only be linked to other nearby genes, but they
can also be linked to molecular markers such as a single
nucleotide changes. This allows an allele of a gene (one that
might cause a disease) to be tracked indirectly just by
following what unique DNA sequences are near it and therefore
tend to be transmitted (associated) with it.

So, if you don’t know what gene causes a particular disease,

you might be able to identify it by finding genomic markers that
are carried by affected individuals. The disease gene is often
linked (nearby) to those markers.
 Unique sequences along a chromosome mark
adjacent alleles

Restriction Fragment Length

Polymorphism/Single Nucleotide
Polymorphism
NNNNNNNNNNNGATCNNNNNNNNNNN~~Gene
NNNNNNNNNNNGACCNNNNNNNNNNN~~Gene*

Micro/Mini Satellite Repeats

NNNNNNNNNNNAGAGAGAGNNNNNNNNNNNNNN~~Gene
NNNNNNNNAGAGAGAGAGAGAGAGAGNNNNNNN~~Gene*
These differences are also the basis for DNA
fingerprinting/forensics
Blue lines represent
microsatellite sequence
markers that have been
identified along human
chromosomes.
In identifying a disease gene
one hopes to find specific
marker sequences that are
usually present in disease
patients but only sometimes
present in unaffected
individuals.
Such associations indicate
the markers are close to the
gene that causes the disease.
Patients consistently carry the
marker because meiotic
recombination has not
separated them from that
gene.
But, its not easy.