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Chapter 7.

Linkage, Crossing Over, & Chromosome Mapping in


Eukaryotes
1. Linkage, Recombination, and Crossing over
2. Chromosome Mapping
3. Cytogenetic Mapping

Chiasmata in the late prophase of the first meiotic


division. These cross-shaped figures are the result of
exchanges between paired chromosomes.

The World's First Chromosome Map


chromosome organization emerged from
a combination of genetic and cytological
studies.
T. H. Morgan laid the foundation for these
studies when he demonstrated that the
gene for white eyes in Drosophila was
located on the X chromosome.
The map was a straight line, and each
gene was situated at a particular point,
or locus, on it
Locus: A fixed position on a
chromosome that is occupied by a given

One night in 1911 Sturtevant,


undergraduate working in Morgan's
laboratory put aside his algebra
homework in order to evaluate some
experimental data.
Before the sun rose the next day, he
had constructed the world's first
chromosome map.

No microscope was powerful enough to see genes,


nor was any measuring device accurate enough to
obtain the distances between them.
In fact, Sturtevant did not use any sophisticated
instruments in his work.
Instead, he relied completely on the analysis of data
from experimental crosses with Drosophila.

1. Linkage, Recombination, and Crossing Over


Sturtevant based his mapping procedure on the
principle that genes on the same chromosome
should be inherited together.
Because such genes are physically attached to the
same structure, they should travel as a unit through
meiosis. This phenomenon is called linkage.
Linkage: A relationship among genes in the same
chromosome. Such genes tend to be inherited
together.

The early geneticists also knew that linkage was not


absolute.
Their experimental data demonstrated that genes on
the same chromosome could be separated as they
went through meiosis and that new combinations of
genes could be formed.
However, this phenomenon, called recombination,
was difficult to explain by simple genetic theory.

One hypothesis was that during meiosis, when


homologous chromosomes paired, a physical
exchange of material separated and recombined
genes.
cytological observation

A crossover point was called a chiasma (plural,


chiasmata), from the Greek word meaning cross.

Exceptions to the Mendelian principle of


independent assortment
Bateson and Punett
sweet peas
two traits, flower color and pollen length
Red flower, long pollen grain are dominant.
9:3:3:1 ???

=803

24.3:1.1:1:7.1

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The chi-square value is enormousmuch greater


than 7.8, which is the critical value for a chi-square
distribution with three degrees of freedom
Consequently, we must reject the hypothesis that
the genes for flower color and pollen grain length
have assorted independently

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Some nonparental progeny do appear, although at


low frequency. Because these progeny indicate that
the alleles of the two genes were recombined in the
F1, they are called recombinants.

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Frequency of recombination as a measure of


linkage intensity
We can use the frequency of recombination to
measure the intensity of linkage between genes.
Genes that are tightly linked seldom recombine,
whereas genes that are loosely linked recombine
often.

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Each phenotype is highlighted by a different color in the


Punnett square. These phenotypes do not appear in a
9:3:3:1 ratio because the genes for flower color and pollen14
length are located on the same chromosome.

Frequency of recombination as a measure of


linkage intensity

The most direct way of estimating the frequency of


recombination is to use a testcross, in which the
individual to be analyzed genetically is crossed to a
homozygote carrying the recessive alleles.

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AA BB individual is crossed to an aa bb individual.


From this cross, the Aa Bb offspring are then
testcrossed to the double recessive parent.
Because the A and B genes assort independently,
the F2 will consist of two classes (Aa Bb and aa bb)
that are phenotypically like the parents in the
original cross, and two classes (Aa bb and aa Bb)
that are phenotypically recombinant.
Furthermore, each F2 class will occur with a
frequency of 25 percent.

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For any two genes, the frequency of recombinant


gametes never exceeds 50 percent.
This upper limit is reached when genes are very far
apart, perhaps at opposite ends of a chromosome.
It is also reached when genes are on different
chromosomes; 50 percent recombination is, in fact,
what is meant when it is said that the genes assort
independently.

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Crosses involving linked genes are usually diagrammed


to show the linkage phasethe way in which the
alleles are arranged in heterozygous individuals

linkage phase : coupling linkage phase


repulsion linkage phase

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Crossing Over as the Physical Basis of


Recombination
Recombinant gametes are produced as a result of
crossing over between homologous chromosomes.
The exchange event occurs during the prophase of
the first meiotic division, when duplicated
chromosomes have paired.
Although four homologous chromatids are present,
forming what is called a tetrad, only two chromatids
cross over at any one point.

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Crossing over as the physical basis of


recombination

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The evidence for crossing over between two


chromatids within a tetrad comes from the study of
certain fungi in the class Ascomycetes,
Saccharomyces cerevisiae.
One important finding from studies with S. cerevisiae
is that crossing over occurs after homologous
chromosomes have duplicated.
An ascus may contain two recombinant and two
nonrecombinant ascospores, each of which has a
different genotype.
This observation can only be explained if crossing
over occurs after chromosome duplication. If it
occurred before duplication, an ascus could never
contain more than two kinds of ascospores.

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A second finding is that only two chromatids are


involved in an exchange at any one point.
However, the other two chromatids may cross over
at a different point.
Thus, there is a possibility for multiple exchanges in
a tetrad of chromatids

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Evidence That Crossing Over Causes Recombination


In 1931 Harriet Creighton and Barbara McClintock
obtained evidence that genetic recombination was
associated with a material exchange between
chromosomes.
Creighton and McClintock studied homologous
chromosomes in maize that were morphologically
distinguishable.
The goal was to determine whether physical
exchange between these homologues was correlated
with recombination between some of the genes they
carried.
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Harriet Creighton and Barbara McClintock:


maize
two forms of chromosome 9
one was normal, and the other had cytological
aberrations at each enda heterochromatic knob at
one end and a piece of a different chromosome at
the other

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Evidence that crossing over causes recombination

Kernel color ( C, colored ; c, colorless )


Kernel texture (Wx, starchy ; wx,
waxy )

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Evidence that crossing over causes recombination

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Chiasmata and the Time of Crossing Over


The cytological evidence for crossing over can be seen
during late prophase of the first meiotic division when
the chiasmata become clearly visible.

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As we might expect, large


chromosomes typically
have more chiasmata than
small chromosomes.
Thus, the number of
chiasmata is roughly
proportional to
x
chromosome length.
When the heat shocks
were administered late in
prophase, there was little
effect, but when they were
given earlier, the
recombination frequency
was changed.

Diplonema of male meiosis


in the grasshopper
Chorthippus parallelus
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Although almost all the DNA is synthesized during the


interphase that precedes the onset of meiosis, a small
amount is made during the first meiotic prophase.
This limited DNA synthesis has been interpreted as
part of a process to repair broken chromatids, which, as
we have discussed,
is thought to be associated with
x
crossing over.

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2. Chromosome Mapping
1) Crossing over as a measure of genetic distance
Sturtevant's fundamental insight was to estimate the
distance between points on a chromosome by
counting the number of crossovers between them.
Points that are far apart should have more
crossovers between them than points that are close
together.
In a statistical sense. The distance between two
points on the genetic map of a chromosome is the
average number of crossovers between them.

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we cannot see
each of the
exchange points
on the
chromosomes
coming out of
meiosis.
Instead, we infer
their existence by
observing the
recombination of
the alleles that
flank them.
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2) Recombination mapping with


a two-point testcross

nonrecombinants
(0) x 0.82
0.18

recombinants
(1) x 0.18 =

- map unit a centiMorgan ( cM ,


M)

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3) Recombination mapping with a three-point


testcross
Determination of the gene order
Three possible gene orders:

cv

(i)sc

ec

cv

(ii)ec

sc

cv

(iii) ec

cv

sc

ec

sc
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Calculation of the distances between genes

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Calculation of the distances between genes

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Sc---9.1---ec---10.5---cv
We can also obtain this estimate by directly calculating the
average number of crossover between these genes:
Noncrossover
classes

Single crossover
classes

( 1and 2 )

( 3,4,5, and 6 )

( 0 ) x 0.805
+
0.196

( 1 ) x 0.195

(1158+1455)/3248

Double crossover
classes
( 7 and 8)
+

( 2) x 0.0006 =
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4) Interference and the coefficient of coincidence


A three-point cross has an important advantage over a
two-point cross: it allows the detection of double
crossovers, permitting us to determine if exchanges in
adjacent regions are independent of each other.
Does a crossover in the region between sc and ec (region
I on the map of the X chromosome) occur independently
of a crossover in the region between ec and cv (region
II)? Or does one crossover inhibit the occurrence of
another nearby?

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one crossover inhibited the occurrence of another


nearby, a phenomenon called interference.
The extent of the interference is customarily measured
by the coefficient of coincidence, c, which is the ratio
of the observed frequency of double crossovers to the
expected frequency:

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in region I on Bridges and Olbrycht's map, the crossover


frequency was (163 + 130 + 1 + 1)/3248 = 0.091, and in
region II, it was (192 + 148 + 1 + 1)/3248 = 0.105.
If we assume independence, the expected frequency of
double crossovers in the interval between sc and cv
would therefore be 0.091 0.105 = 0.0095.

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- observed frequency of double crossovers


2 / 3248 = 0.0006
- expected frequency of double crossovers
crossover frequency of region I x crossover
frequency
of region :
( 0.091 ) x (0.105) = 0.0095
Coefficient of coincidence, C
C = observed frequency of double crossover
expected frequency of double crossover
= 0.0006 / 0.0095
= 0.063
Interference, I
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I = 1- C = 0.937

5) Recombination frequency and genetic map distance


A discrepancy between map distance and percent
recombination.

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As an example, let us consider the genes at the ends


of Bridges and Olbrycht's map of the X chromosome;
sc, at the left end, was 66.8 cM away from f, at the
right end.
However, the frequency of recombination between sc
and f was 50 percentthe maximum possible value.
Using this frequency to estimate map distance, we
would conclude that sc and f were 50 map units apart.

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Multiple crossovers may occur between widely


separated genes, and some of these crossovers may
not produce genetically recombinant chromosomes

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Relationship between frequency of


recombination and genetic map distance

Two-strand double crossovers produce all parental chromosomes

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Relationship between frequency of


recombination and genetic map distance

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Relationship between frequency of


recombination and genetic map distance

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This second example shows that a double crossover


may not contribute to the frequency of recombination,
even though it contributes to the average number of
exchanges on a chromosome.
A quadruple crossover would have the same effect.
These and other multiple exchanges are responsible
for the discrepancy between recombination frequency
and genetic map distance.

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For values less than 20 cM, there is approximately a linear


relationship between percent recombination and map
distance; for values greater than 20 cM, the percent
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recombination underestimates the map distance.

6) Chiasma frequency and genetic map


distance
Each chiasma is thought to represent the resolution of
a crossover that occurred earlier in prophase.
Thus, by counting chiasmata, we should be able to
estimate the average number of crossovers occurring
on a chromosome, which we can then use as an
estimate of genetic map length.
Let's suppose, for example, that in a group of 100
cells going through meiosis, 5 cells show five
chiasmata in a particular pair of chromosomes, 15
show four chiasmata in this pair, 15 show three, 30
show two, 25 show one, and 10 show none
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This translates into an average of 1.07 chiasmata per


chromatid because each chiasma affects only two of the57
four chromatids in the chromosome pair

3. Cytogenetic Mapping
Recombination mapping allows us to determine the
relative positions of genes by using the frequency of
crossing over as a measure of distance.
However, it does not allow us to localize genes with
respect to cytological landmarks, such as bands, on
chromosomes.
This kind of localization requires a different procedure
that involves studying the phenotypic effects of
chromosome rearrangements, such as deletions and
duplications.
The map positions of those genes can be tied to
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locations on the cytological map of a chromosome. This

cytologically defined deletion (or deficiency, usually symbolize


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We can also use duplications to determine the


cytological locations of genes.
We look for a duplication that masks the phenotype of
a recessive mutation.

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The basic principle in deletion mapping is that a


deletion that uncovers a recessive mutation must lack
a wild-type copy of the mutant gene.
This fact localizes that gene within the boundaries of
the deletion.
The basic principle in duplication mapping is that a
duplication that covers a recessive mutation must
contain a wild-type copy of the mutant gene.
This fact localizes that gene within the boundaries of
the duplication.
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4. Genetic Distance and Physical


Distance
We expect that long chromosomes should have more
crossovers than short ones and that this relationship
will be reflected in the lengths of their genetic maps.
For the most part, our assumption is true; however,
within a chromosome some regions are more prone to
crossing over than others.
Thus, distances on the genetic map do not correspond
exactly to physical distances along the chromosome's
cytological map
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Crossing over is less likely to occur near the ends of a


chromosome and also around the centromere;
consequently, these regions are condensed on the
genetic map.
Other regions, in which crossovers occur more
frequently, are expanded.

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1) Genetic distance and physical distance

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Even though there is not a uniform relationship


between genetic and physical distance, the genetic
and cytological maps of a chromosome are colinear;
that is, particular sites have the same order.
Recombination mapping therefore reveals the true
order of the genes along a chromosome. However, it
does not tell us the actual physical distances between
them.

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2) Detecting linked loci by pedigree analysis


To detect and analyze linkage in humans, geneticists
must collect data from pedigrees.
Often these data are limited or incomplete, or the
information they provide is ambiguous.
Challenge
Today, modern molecular methods permit researchers
to analyze the inheritance of dozens of different
markers in the same set of pedigrees.
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In addition, breakthroughs in cytogenetics and cell


culture techniques have allowed geneticists to tie
these maps to individual chromosomes and to localize
genes to specific regions within them.

The linkage relationships that are easiest to study in


human beings are those between genes on the X
chromosome.
Determining linkage between autosomal genes is
much more difficult. The human genome has 22
different autosomes, and a gene that does not show Xlinkage could be on any one of them.
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linkage between the gene controlling the ABO blood groups


abnormal nails and kneecaps

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The woman in generation II must represent a new


occurrence of the NPS1 mutation. Neither of her
parents nor any of her 11 siblings showed the nailpatella phenotype.
Among the five individuals who showed the nailpatella syndrome in this pedigree, all but one (III-6) of
them had blood type B.
NPS1 is linked to the B allele

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If we assume this inference to be correct, then the


woman in generation II must have the genotype NPS1
B/ + O;. Her husband's genotype is clearly + O/ + O.

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The mating is a testcross. However, only 3 (III-3, III-6,


III-12) of their 10 children were recombinants; the other
7 were nonrecombinants. Thus, we can estimate the
frequency of recombination, 3/10 = 30 percent.
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However, this estimate does not use all the information


in the pedigree. the information from the couples' three
grandchildren, only one (IV-1) of whom was a
recombinant. Altogether, then, 3 + 1 = 4 of the 10 + 3
= 13 offspring in the pedigree were recombinants.
Thus, we conclude that the frequency of recombination
between the NPS1 and ABO loci is 4/13 = 31 percent. 74

The first localization of a gene to a specific human


autosome came in 1968, when R. P. Donahue and
coworkers demonstrated that the Duffy blood group
locus, denoted FY, is on chromosome 1.
This demonstration hinged on the discovery of a
variant of chromosome 1 that was longer than normal.
Pedigree analysis showed that in a particular family,
this long chromosome segregated with specific FY
alleles.

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Somatic-Cell Techniques for assigning genes to


chromosomes
Until the 1970s, assigning linkage mapsand
individual genesto human chromosomes was an
exceedingly difficult task.
Two technical advances made this task much easier.
First, new dyes allowed individual chromosomes to be
distinguished inside cells.
Second, researchers discovered ways of culturing
somatic cells that had been formed by fusing cells
from different species.
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hybrid cells are formed by mixing human cells with


the cells of another animal, usually a rodent such as a
mouse or a hamster.
The cells are mixed in culture in the presence of
polyethylene glycol, a chemical that induces the
fusion of cell membranes.
If a human cell fuses with a rodent cell, the resulting
cell will have the chromosomes of both species.
However, when such a cell divides, the human
chromosomes are randomly lost.
After many cycles of division, the chromosome
content of the cultured cells stabilizes

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One problem with this procedure is that fusion takes


place between cells of the same species as well as
between cells of different species.
To select for interspecific hybrid cells, researchers
grow the mixed cultures on a medium in which only
such hybrid cells survive.

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In addition to nutritive materials, the preferred


medium contains three chemicalshypoxanthine,
aminopterin, and thymidine (HAT medium).
Cells deficient for either of two enzymes, thymidine
kinase (TK) or hypoxanthine phosphoribosyl
transferase (HPRT), cannot grow in HAT medium.
Thus, if human cells that are TK (that is, deficient in
thymidine kinase) and mouse cells that are HPRT
(that is, deficient in hypoxanthine phosphoribosyl
transferase) are placed in HAT medium, neither type
will grow.
If the two types of cells (TK+, HPRT+) fuse, hybrids
will grow in HAT medium because the fused cells
compensate for each other's defects.

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Recombination and Evolution


Evolutionary Significance of Recombination
-In the sexual organism, the two mutations can be
recombined to produce a strain that is better than
either of the single mutants by itself.

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Suppression of Recombination by Inversions


-Crossing over is usually inhibited near the
breakpoints of a rearrangement in heterozygous
condition, probably because the rearrangement
disrupts chromosome pairing.

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The net effect of this


chromatid loss is to suppress
recombination between
inverted and noninverted
chromosomes in
heterozygotes.
Acentric fragment,
a dicentric chromatid bridge
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Euploid products result from


crossing over between inverted
and noninverted chromatids
Two crossovers occur within the
inverted region.
Both of the crossovers must
involve the same two
chromatidsa so-called twostrand double exchange.
If they involve different
chromatids, the products of the
exchanges will be aneuploid.
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Geneticists have exploited the recombinationsuppressing properties of inversions to keep alleles of


different genes together on the same chromosome.
Balancers: marked inversion chromosomes
they allow a mutant chromosome to be kept in
heterozygous condition over the inversion.

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Genetic Control of Recombination


Studies with several organisms, including yeast and
Drosophila, have demonstrated that recombination
involves the products of many genes.
One curious phenomenon, which no one has yet
explained, is that there is no crossing over in
Drosophila males.

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