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Chapter 14
Genetic Mapping in
Eukaryotes
Copyright © 2010 Pearson Education Inc.
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A chiasma (plural, chiasmata) is the site on the
homologous chromosomes where crossover
occurs.
Crossing-over is the reciprocal exchange of
homologous chromatid segments, involving the
breaking and rejoining of DNA.
Crossing-over is also the event leading to
genetic recombination between linked genes in
both prokaryotes and eukaryotes.
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Genes on nonhomologous chromosomes assort
independently, but genes on the same chromosome
(syntenic genes) may instead be inherited together
(linked) and belong to a linkage group.
Classical genetics analyzes the frequency of allele
recombination in progeny of genetic crosses.
◦ a. New associations of parental alleles are recombinants,
produced by genetic recombination.
◦ b. Testcrosses determine which genes are linked, and a linkage
map (genetic map) is constructed for each chromosome.
◦ c. Genetic maps are useful in recombinant DNA research and
experiments dealing with genes and their flanking sequences
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Current high-resolution maps include both gene
markers from testcrosses and DNA markers
composed of genomic regions that differ
detectably between individuals.
Genomic sequencing allows determination of exact
positions of genes. These physical maps use
molecular tools, rather than data from cross-over
studies.
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Morgan’s Experiments with Drosophila
Both the white eye gene (w) and a gene for miniature
wings (m) are on the Drosophila X chromosome.
Crossed a female white miniature (w m/w m) with a
wild-type male (w+m+/Y).
◦ a. In the F1, all males were white-eyed with miniature wings (w
m/Y), and all females were wild type for both eye color and
wing size (w+m+/w m).
◦ b. F1 interbreeding is the equivalent of a testcross for these Xlinked genes, since the male is hemizygous recessive, passing
on recessive alleles to daughters and no X-linked alleles at all
to sons.
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In the F2, the most frequent phenotypes for both
sexes were the phenotypes of the parents in the
original cross (white eyes with miniature wings and
red eyes with normal wings).
Nonparental phenotypes (white eyes with normal
wings or red eyes with miniature wings) occurred
in about 37% of the F2 flies.
Well below the 50% predicted for independent
assortment, this indicates that nonparental flies
result from recombination of linked genes.
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During meiosis, alleles of some genes assort
together because they are near each other on the
same chromosome.
Recombination occurs when genes are
exchanged between the X chromosomes of the F1
females.
Crossing-over occurs at the four-chromatid stage of prophase
I in meiosis. Each crossover event involves two of the four
chromatids. All chromatids may be involved in crossing-over,
as chiasmata form along the aligned chromosomes.
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Stern worked with two linked
gene loci in Drosophila
melanogaster. The linked loci
were car and B:
◦ a. The car (carnation) gene is
recessive. Homozygotes have
carnation-colored eyes, rather than
wild-type red. The car locus is near
the “left” end of the X chromosome.
◦ b. The B (bar-eye) gene is
incompletely dominant.
Homozygotes (B/B) have a barshaped eye rather than wild-type
nonbar (round). Heterozygotes
(B/B+) have a wide-bar (kidneyshaped) eye. The B locus is farther
from the “left” end of the X
chromosome (thus nearer the
centromere) than is the car locus.
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Male parents carried recessive alleles for
both eye color (car) and eye shape (1) on a
single X chromosome (car B+/Y). Phenotype
is carnation, nonbar eyes (wild type).
Female parent carried two abnormal and
cytologically distinct X chromosomes, with a
genotype of car+ B+/car B, and a phenotype
of kidney-shaped red eyes.
i. One X chromosome had a translocated fragment of Y chromosome. It
carried the wild-type alleles (car+ B+, red, and nonbar) for both traits.
ii. The second X chromosome had lost a region by translocation to
chromosome 4. This chromosome was visibly shorter than a normal X
chromosome. Its alleles were the two mutants, car and B.
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Gamete formation would produce two types in males, X with both
recessive alleles, and Y with neither of the alleles.
Females produce four types, two nonrecombinant and two recombinant.
A Punnett square shows the segregation of alleles.
Cytological examination of progeny showed:
i. Both males and females with nonbar
carnation eyes had a normal X chromosome,
along with a second normal X in females, or
a Y in males.
ii. Female flies with wide-bar red eyes and
males with bar red eyes had a short X
chromosome with the Y translocation, along
with a normal X or Y.
This confirmed that physical crossing-over
between chromosomes results in genetic
recombination.
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Genetic recombination experiments can be
used in genetic (or linkage) mapping.
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Linked genes are used for mapping. They are
found by looking for deviation from the
frequencies expected from independent
assortment.
A testcross (one parent is homozygous recessive)
works well for analyzing linkage:
◦ a. If the alleles are not linked, and the second parent is
heterozygous, all four possible combinations of traits will
be present in equal numbers in the progeny.
◦ b.A significant deviation in this ratio (more parental and
fewer recombinant types) indicates linkage.
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In an individual heterozygous at two loci,
there are two arrangements of alleles:
◦ a. The cis (coupling) arrangement has both wildtype alleles on one homologous chromosome and
both mutants on the other (e.g., w+m+ and w m).
◦ b.The trans (repulsion) arrangement has one
mutant and one wild type on each homolog (e.g.,
w+m and w m+).
◦ c. A crossover between homologs in the cis
arrangement results in a homologous pair with the
trans arrangement. A crossover between homologs
in the trans arrangement results in cis homologs.
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Drosophila crosses showed that crossover frequency
for linked genes (measured by recombinants) is
characteristic for each gene pair. The frequency stays
the same, whether the genes are in coupling or in
repulsion.
◦ a. Morgan and Sturtevant (1913) used recombination
frequencies to make a genetic map.
 i. A 1% crossover rate is a genetic distance of 1 map unit (mu). A
map unit is also called a centimorgan (cM).
 Ii. Geneticists use recombination frequency as a way to estimate
crossover frequency. It is not an exact measure, however.
 Iii. The farther apart the two genes are on the chromosome, the
more likely it is that crossover will occur between them, and
therefore the greater their crossover frequency.
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Based on crosses in Drosophila involving the three
sex-linked genes:
◦ i.w gives white eyes.
◦ ii.m gives miniature wings.
◦ iii.y gives yellow body.
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The crosses gave the following recombination
frequencies:
◦ i.w X m was 32.7.
◦ Ii.w X y was 1.0.
◦ Iii.m X y was 33.7.
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Map is therefore:
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With autosomal recessive alleles,
when a double heterozygote is
testcrossed, four phenotypic
classes are expected. If the genes
are linked, the two parental
phenotypes will be about equally
frequent and more abundant
than the two recombinant
phenotypes
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Mapping of genes with other mechanisms of inheritance is
also done with two-point testcrosses:
◦ a. For X-linked recessives, a female double heterozygote (a+b+/a b)
is crossed with a male hemizygous for the recessive alleles (a b/Y).
◦ b. For either X-linked case, it is possible to cross the females with
males of any type. As long as only male progeny are analyzed, the
father’s X will be irrelevant.
◦ c. Phenotypes obtained in any of these crosses will depend on
whether the alleles are arranged in coupling (cis) or repulsion
(trans).
◦ d. Recombination frequency is used directly as an estimate of map
units. The same principles of mapping apply in using both gene
markers and DNA markers.
 i.The measure is more accurate when the alleles are close together.
 Ii.Scoring large numbers of progeny increases the accuracy.
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Mapping in all types of organisms shows genes arranged
with a 1:1 correspondence between linkage groups and
chromosomes.
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A genetic map is generated from estimating the crossover
rate in a particular segment of the chromosome. It may not
exactly match the physical map, because crossover is not
equally probable at all sites on the chromosome.
Recombination frequency is also used to predict progeny in
genetic crosses. For example, a 20% crossover rate between
two pairs of alleles in a heterozygote (a+b+/a b) will give
10% gametes of each recombinant type (a+b and a b+).
A recombination frequency of 50% means that genes are
unlinked. There are two ways in which genes may be
unlinked:
◦ a. They may be on separate chromosomes.
◦ b. They may be far apart on the same chromosome.
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If the genes are on the same chromosome,
multiple crossovers can occur. The further apart
two loci are, the more likely they are to have
crossover events take place between them. The
chromatid pairing is not always the same in
crossover, so that 2, 3, or 4 chromatids may
participate in multiple crossover.
To determine whether the genes are on the same
chromosome, or on different ones, other genes
in the linkage group may be mapped in relation
to a and b and used to deduce their locations.
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Typically, geneticists design
experiments to gather data
on several traits in one
testcross. An example of a
three-point testcross would
be p+r+j+/p r j X p r j/p r j
In the progeny, each gene
has two possible phenotypes.
For three genes there are (2)3
= 8 expected phenotypic
classes in the progeny.
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The order of genes on the chromosome can be
deduced from results of the cross. Of the eight
expected progeny phenotypes:
◦ a. Two classes are parental (p+r+j+/p r j and p r j/p r j) and
will be the most abundant.
◦ b.Of the six remaining phenotypic classes, two will be
present at the lowest frequency, resulting from apparent
double crossover (p+r+j/p r j and p r j+/p r j). This
establishes the gene order as p j r .
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Cross data is organized to
reflect the gene order, and
in this example the region
between genes p and j is
called region I, and that
between j and r is region II.
Recombination frequencies
are now calculated for two
genes at a time. It includes
single crossovers in the
region under study, and
double crossovers, since
they occur in both regions.
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Recombination frequencies are used to position genes on
the genetic map (each 1% recombination frequency = 1
map unit) for the chromosomal region.
Recombination frequencies are not identical to crossover
frequencies and typically underestimate the true map
distance.
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Double crossovers do not occur as often as expected from
the observed rate of single crossovers. Crossover appears to
reduce formation of other chiasmata nearby, producing
interference. Interference = 1 is total interference, with no
other crossovers occurring in the region.
The coefficient of coincidence expresses the extent of
interference.
◦ a. Interference = 1 minus the coefficient of coincidence. The values
are inversely related.
◦ b. A value of 1 means the number of double crossovers that occurs
is what would be predicted on the basis of two independent
events, and there is no interference.
◦ c. A value of 0 means that none of the expected crossovers
occurred, and interference is total.
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Recombination frequency generally
underestimates the true map
distance:
◦ a. Double crossovers between two loci
will restore the parental genotype, as
will any even number of crossovers.
These will not be counted as
recombinants, even though crossovers
have taken place.
◦ b. A single crossover will produce
recombinant chromosomes, as will any
odd number of crossovers. Progeny
analysis assumes that every
recombinant was produced by a single
crossover.
◦ c. Map distances for genes that are less
than 7 mu apart are very accurate. As
distance increases, accuracy declines
because more crosses go uncounted.
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Mapping functions are mathematical formulas used
to define the relationship between map distance
and recombination frequency. They are based on
assumptions about the frequency of crossovers
compared with distance between genes.
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Gene or DNA mapping based on crossover
assumes that crossover sites are random. This is
not entirely true, because hot spots and cold spots
for crossover occur on chromosomes. Nonrandom
distribution of crossing-over limits accuracy of
mapping by this method.
DNA sequence is the best physical map.
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The lod Score Method for Analyzing Linkage of Human
Genes
◦ The lack of suitable human pedigrees showing segregation of
defined linked traits makes mapping autosomes especially
difficult, and so usually the lod (logarithm of odds) score
method is used for statistical analysis of pedigree data.
◦ a. A lod score compares the expected distributions of traits if
they are linked, and if they are not linked.
◦ b. The lod score is the log10 of the ratio of the two probabilities.
The higher the lod score, the closer the two genes.
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The map distance for linked markers is computed from
the recombination frequency given by the highest lod
score, by solving lod scores for a range of proposed
map units.
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The Human Genome Project has used two
approaches:
◦ a. The mapping approach, based on landmarks used to
construct physical maps that are correlated with
sequencing data. This approach was important at the
beginning of the genome project.
◦ b.The shotgun approach, sequencing random fragments
and then using computers to assemble the genome based
on overlaps between fragments. This is the technique now
routinely in use.
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Human genetic mapping was revolutionized by:
◦ a. Discovery of many polymorphic DNA markers.
◦ b. Development of molecular tools to type the markers
(determining molecular phenotypes). PCR is an important
example.
◦ c. Short tandem repeats (STRs), also called microsatellites, are
2–6-bp repeats that form polymorphic loci that can be detected
by PCR.
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Hundreds of markers may be typed in a given cross,
and computer algorithms are used to determine
linkage relationships.
A high-density human genetic map was completed in
1994.
◦ a. A consortium of laboratories worked on the same set of DNA
samples (mapping panel), so their data could be combined.
◦ b. Localized 5,840 loci with resolution of about 0.7 mu.