LINKAGE, CROSSING-OVER, AND

GENE MAPPING IN EUKARYOTES

• Discuss the scientific contributions of
Morgan and others that led to the
discovery of genetic linkage
• Describe how chromosomes exchange
genetic material via crossing-over of
chromosomes during meiosis, and how
this promotes genetic variability
• Construct genetic maps from crossover
data
 Discovery of Genetic Linkage

• Genes on non-homologous chromosomes

assort independently

• Genes on the same chromosome may

instead be inherited together (linked), and
belong to a linkage group
• Classicalgenetics analyzes the frequency
of allele recombination in progeny of
genetic crosses

 New associations of parental alleles are

recombinants, produced by genetic
recombination

 Testcrosses determine which genes are

linked and a linkage map (genetic map) is
constructed for each chromosome

 Genetic maps are useful in recombinant DNA

research and experiments dealing with
genes and their flanking sequences
• Current high resolution maps include
both:

Gene markers from testcrosses

DNA markers composed of genomic

regions that differ detectably between
individuals
 Linkage in the Sweet Pea
• Bateson, Saunders, and Punnett in 1905
found exceptions to the law of
independent assortment in their studies
of pea plant

• They crossed two true-breeding strains:

one that produced purple flowers and
long pollen and another that produced red
flowers and round pollen
 Linkage in the Sweet Pea
• Expected results of F1 X F1 cross if genes
for flower color & pollen size were to assort
independently

• F1 generation: diploid, Genotype Pp Ll

Phenotype Purple flowers, long pollen

• Expected genotypes of possible F1 gametes

haploid ¼ PL, ¼ Pl, ¼ pL, ¼ pl
• Expected phenotypic ratios of F2 generation
Purple long Purple round Red long Red round
9/16 3/16 3/16 1/16
% 56.25 18.75 18.75 6.25
• Observed results:
69.5 5.6 5.6 19.3
• Observed results indicated partial linkage of
genes. Dominant purple and long characters
occurred together more often than predicted
• F1 haploid gametes
44% PL 6% Pl 6% pL 44% pl
 Morgan’s Linkage Experiments
with Drosophila
• Both the white eye gene (w) and a gene for
miniature wing (m) are on the X chromosome
• Morgan (1911) crossed a female white
miniature (w m/w m) with a wild-type male
(w+ m+/Y)
• In the F1, all males are white eyed with
miniature wings and all females are wild-type
for both eye color and wing size
• Morgan concluded that during meiosis, alleles
of some genes do not assort independently
but assort together because they lie near one
another on the same chromosome
• F1 interbreeding is the equivalent of a
testcross for these X-linked genes, since
the male is hemizygous recessive
• In the F2, the most frequent phenotypes
for both sexes are the phenotypes of the
parents in the original cross (white eyes
with miniature wings, and red eyes with
normal wings)
• Non-parental phenotypes occurred in
about 37% of the F2 flies below the
predicted 50% for independent
assortment
• This indicates non-parental flies result
from recombination of linked genes
 Morgan’s experimental crosses of white-eye and
miniature-wing variants ofDrosophila melanogaster
Morgan’s Linkage Experiments
with Drosophila
• Morgan proposed that:

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
 TERMINOLOGY
•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
Mechanism of Crossing-Over
 GENE RECOMBINATION AND
THE ROLE OF CHROMOSOMAL
EXCHANGE
 Corn Experiments
• Creighton and McClintock (1931) performed
crosses with corn plants and found cytological
evidence that crossing-over occurs during
meiosis and is associated with the physical
exchange of parts between homologous
chromosomes
• The study used a corn strain heterozygous
for two genes on chromosome 9
• One gene determines seed color (C for
colorled seeds, c for colorless)
• The other gene is involved in starch
synthesis:
Wild-type allele (Wx) produces amylose,
and amylose + amylopectin = normal
starch
Waxy mutant (wx) lacks amylose
• In this corn strain, the appearance of each
chromosome 9 homolog correlated with its
genotype
Evidence of the association of gene recombination
with chromosomal exchange in corn
 Drosophila Experiments
• Shortly after Creighton and McClintock
published their results, Stern reported
identical results for experiments done
with Drosophila
• He reported on two linked gene loci
• From experiments such as these, it
became apparent that genetic
recombination results from physical
crossing-over between chromosomes
Stern’s experiment to
demonstrate the
relationship between
genetic recombination
and chromosomal
exchange in Drosophila
melanogaster
 Crossing-Over at the Tetrad
Stage of Meiosis
• Crossing-over occurs at the four-chromatid
(tetrad) stage in prophase I during meiosis
• In Neurospora crassa (orange bread mold)
forms eight haploid spores. Their
arrangement in the ascus refects the
orientation of chromatids in the metaphase
tetrad of meiosis
• To determine when crossing-over occurs,
crosses were made between haploid
Neurospora strains of different mating types
(A and a)
Life cycle of
the haploid,
mycelial-
form fungus
Neurospora
crassa
Experiment
showing that
crossing-over
occurs at the
four-chromatid
stage of meiosis
LOCATING GENES ON CHROMOSOMES:
MAPPING TECHNIQUES
1. Detecting Linkage Through Testcrosses
2. Gene Mapping by Using Two-Point
Testcrosses
3. Generating a Genetic Map
4. Double Crossovers
5. Three Point Crosses
 Detecting Linkage Through
Testcrosses
• To construct a genetic map or linkage
map, the genes in question must be
linked, or located on the same
chromosome

• To test for linkage, a testcross is used

• Testcrosses are between one individual

of unknown genotype and a
homozygous recessive individual
 Detecting Linkage Through
Testcrosses
• If two genes are not linked, a testcross
should show a 1:1:1:1 phenotypic ratio

• Statistically significant deviations from

the expected results indicate that the
genes are linked and have recombined
(chi-square test)

• Two-linked genes→ too many parental

types and too few recombinant types
Testcross to show that
two genes are linked
 CONCEPT OF A GENETIC MAP
• In an individual heterozygous at two
loci, there are two arrangements of
alleles:
 Cis (coupling) arrangement has both
wild-type alleles on one homologous
chromosome and both mutants on the
other (w+ m+ and w m)
 Trans (repulsion) arrangement has one
mutant and one wild-type on each
homolog (w+ m and w m+)
•A crossover between homologs in the cis
arrangement results in a homologous pair with
the trans arrangement and vice versa
• Frequency of recombinants is the same,
regardless of how the alleles of the two genes
involved are arranged relative to each other on
the homologous chromosomes
• Geneticists use recombination frequencies to
make a genetic map
• A 1% crossover rate is a genetic distance of 1
map unit (mu) = 1 centimorgan (cM)
• Genetic distances are additive
 Gene Mapping Using Two-Point
Testcrosses
• The percentage of recombinants resulting from
crossing-over is used as a measurement of the
genetic distance between two linked genes
• In all cases, a two-point testcross should yield
a pair of parental types that occur with equal
frequency and a pair of recombinant types that
also occur equally
• The value for the percentage of recombinants
(# recombinant/total # of progeny X 100) is
usually directly converted into map units
 TWO-POINT TESTCROSSES
• Autosomal Recessive Test-cross
a+ b+/a b X a b/a b Homozygous Recessive
• Autosomal Dominant WT alleles are
recessive
A B/A+ B+ X A+ B+/A+ B+
• X-Linked Recessive
a+ b+//a b X a b/
• X-Linked Dominant
A B//A+ B+ X A+ B+/
 Generating a Genetic Map
• A genetic map can be constructed by
estimating the number of times a
crossover event occurred in a particular
segment of the chromosome

• The map distance between two genes is

based on the frequency of
recombination between the two genes
• The recombination frequency between genes
in chromosomes can be computed as the
percentage of progeny showing the
reciprocal recombinant phenotypes

• The closer the recombination frequency

paralleles the crossover frequency, the
closer the genes are

• Multiple crossovers may result: use product

rule to calculate its probability
ex: p= 0.2 for one crossover
p= 0.2 X 0.2 = 0.04 of double crossover
The relationship
between crossing-over
and map distance
• For any testcross, the percentage of
recombinants cannot exceed 50%
• Independent assortment → equal # of
recombinants and parents = 50%
• Unlinked genes → 50% recombination
The two genes are on different
chromosomes
The two genes are on the same
chromosome but are so far apart → map
other genes in the linkage group to
determine whether the former genes are
on the same or different chromosome
 DOUBLE CROSSOVERS
• When the distance between two genes
on a chromosome increases > 10 mu,
the incidence of multiple crossovers
causes the recombination frequency to
be an underestimate of the crossover
frequency and map distance

• The effects of multiple crossovers can

be corrected to provide a more accurate
estimate of map distance
Progeny of single and double crossovers
 THREE POINT CROSSES
• One way to overcome the problem of
genetic mapping when multiple
crossover events occur between linked
genes is to perform a three-point
testcross, which involves three genes
on a short region of the chromosome

• In a three-point testcross, a triple

heterozygous is crossed with a
homozygous recessive for all three
genes
Consequences of a double crossover in a
triple heterozygote for three linked genes
Three-point mapping,
showing the testcross used
and the resultant progeny
Rearrangement of the three genes to p j r
Rewritten form of
the testcross and
testcross progeny
based on the
actual gene order
pjr
 Genetic map of the p-j-r region of the
chromosome computed from the
recombination data of the previous slide
THE END