UNDERSTANDING

LINKAGE,
AND GENETIC
MAPPING
 INTRODUCTION

• Each species of organism must contain

hundreds to thousands of genes
– Yet most species have at most a few dozen
chromosomes
• Therefore, each chromosome is likely to
carry many hundred or even thousands of
different genes
– The transmission of such genes will violate
Mendel’s law of independent assortment
LINKAGE AND CROSSING
OVER
• In eukaryotic species, each linear
chromosome contains a long piece of DNA
– A typical chromosome contains many hundred
or even a few thousand different genes
• The term linkage has two related meanings
– 1. Two or more genes can be located on the
same chromosome
– 2. Genes that are close together tend to be
transmitted as a unit
• Chromosomes are called linkage groups
– They contain a group of genes that are linked together

• The number of linkage groups is the number of

types of chromosomes of the species
– For example, in humans
• 22 autosomal linkage groups
• An X chromosome linkage group
• A Y chromosome linkage group

• Genes that are far apart on the same chromosome

may independently assort from each other
– This is due to crossing-over
 Crossing Over May Produce Recombinant
Phenotypes

• In diploid eukaryotic species, linkage can be altered

during meiosis as a result of crossing over

• Crossing over
– Occurs during prophase I of meiosis at the bivalent stage
– Non-sister chromatids of homologous chromosomes
exchange DNA segments

• Figure 5.1 illustrates the consequences of crossing

over during meiosis
 The haploid cells contain
the same combination of
alleles as the original
chromosomes

The arrangement of linked

alleles has not been altered

Figure 5.1
 These haploid cells contain a
combination of alleles NOT
found in the original
chromosomes
This new combination of
These are alleles is a result of
termed genetic recombination
parental or These are termed
non- nonparental or recombinant
recombinant cells
cells
 Bateson and Punnett Discovered Two
Traits That Did Not Assort Independently
• In 1905, William Bateson and Reginald Punnett
conducted a cross in sweet pea involving two
different traits
– Flower color and pollen shape
• This is a dihybrid cross that is expected to yield a
9:3:3:1 phenotypic ratio in the F2 generation
– However, Bateson and Punnett obtained surprising
results
Independent Assortment?
Hypothesis for linkage between genes for flower colour and pollen length in sweet peas
Test cross result
Linkage Phases
A much greater proportion
of the two types found in
the parental generation
 Crossing Over and Mapping

• Linkage without crossing over creates only parental (noncrossover)

gametes.

• Linkage with crossing over creates parental gametes and recombinant

(crossover) gametes.

• Interlocus distance is proportional to the degree of crossing over

between.
– Little or no crossing over in close genes.
– Frequent, even multiple crossovers between distant genes.

• Chromosome map, determined from recombination rates, indicates

relative locations of genes on a chromosome.
No Linkage: Independent Assortment
Linkage without Recombination
Linkage with Recombination
YyRr X yyrr

1:1:1:1
 Complete Linkage: P1 Cross

bwhv  bw hv
P1 : 
 
bwhv bw hv

In complete bwhv 
linkage only F1: 
parental bw hv
gametes form
 Complete Linkage: F1 Cross

Not 9:3:3:1 phenotypic ratio! Not 1:1:1:1 testcross ratio!

1:2:1  (complete) linkage ratio 1:1 testcross ratio w/linkage
 Linkage Ratio
• The F2 phenotypic ratio unique to two linked genes in cross of double
heterozygotes.

• If completely linked, should be

– 1:2:1 for F1 X F1
– 1:1 for F1 X test cross parent

• Linkage group - group of genes which show linkage; in theory = N (the

haploid number).
 II. Morgan and Crossing Over
• Morgan discovered crossing over when studying two genes on X
chromosome in Drosophila.

• Morgan proposed that the chiasmata visible on chromosomes were regions

of crossing over.

• Occurs between nonsister chromatids.

Crosses of Two X-linked genes

Expect only parental types if no crossing over occurs

- Confirm this for yourself with a Punnett square.
 Morgan’s Interpretation
• Recombination was caused by linear arrangement of genes and
crossing over.

• Frequency of recombination was determined by distance between

genes:

– y and w recombination rate = 1.3%

– w an m recombination rate = 37.2%
– Therefore y and w were closer together on the chromosome, while
w an m are farther apart.
 Sturtevant and Mapping
• Sturtevant, Morgan’s undergraduate student, discovered frequency of
crossing over between each pair of the 3 genes:

– yellow, white 0.5%

– white, miniature 34.5%
– yellow, miniature 35.4%

Do you see a pattern?

 Sturtevant’s Interpretation
• Sturtevant reasoned that recombination frequencies were additive, so
order of genes on chromosome was yellow-white-miniature.
You only see recombination when it occurs
between the genes you are watching!
 Single Crossovers: Non-crossover
(Parental) and Crossover (Recombinant)
Gametes

What is the maximum % recombination?

 Map Units
• One map unit (centimorgan, cM) = 1% recombination between two
genes
– yellow and white are 0.5 cM apart
– yellow and miniature are 35.4 cM apart
– white and miniature are (35.4-0.5) = 34.9 cM apart

• In Drosophila, crossing over occurs only in females, never in males.

 III. Three-Point Mapping
• You can add % recombination between two genes to find the order of
genes pretty well.

• But the only way to be sure of the order of three genes is by Three-
Point Mapping, which considers 3 genes at once.

• You look for rare double-crossover events, and that is the clue to the
gene order.
Double Crossovers
 Probability of Double Crossovers
• Equals product of each of their individual probabilities:
– if PAxB = 0.20 and PBxC = 0.30 then
– PAxBxC = (0.20)(0.30) = 0.06 = 6 %

• Criteria for 3-point mapping cross:

– Crossover gametes heterozygous at all loci
– Genotypes can be determined from phenotypes
– Sufficient numbers for representative sample
In D. melanogaster, cherub wings(ch), black body(b), and cinnabar eyes(cn),
result from a recessive allele that are all located on Chromosome 2.
A homozygous wild-type fly was mated with a cherub, black and cinnabar fly
and the resulting F1 females were test crossed with a cherub, black
and cinnabar males. Following progeny were produced from the test cross
Coefficient of Coincidence= Observed DCO frequency/ Expected DCO
 3-Point Mapping in Drosophila
• Cross a y ec w female with
• NCO:
wildtype male to get triply
heterozygous mutant female y ec w 4685
and triply hemizygous mutant + ++ 4759 94.44%
male. • SCO:
• Cross the F1 and examine the y++ 80
F2 phenotypes: + ec w 70 1.50%
– NCO: noncrossover y+w 193
– SCO: single crossover (2 + ec + 207 4.00%
types) • DCO:
– DCO: double crossover
y ec + 3
++w 3 0.06%
• Total: 1000 100%
3-point Mapping Explanation
To Deduce the Order from a 3-Point Cross:
Method 1
1. Group the 8 phenotypic groups into 4 reciprocal pairs.
2. The Non-crossover (NCO) pair is the largest group. The Double
crossover (DCO) pair is the smallest group.
3a. Note which gene “switches” from the parental arrangement in DCO
(present on its own) - that one is in the middle.
 Possible Orders of 3 Genes

• If yellow were in the middle, yellow phenotype would show up in DCO.

• If echinus were in the middle, echinus phenotype would show up in DCO.

• white is actually in the middle since white phenotype shows up in actual

DCO data.
To Deduce the Order from a 3-Point Cross:
Method 2
3b. Assume one of the 3 possible gene orders and work the problem. If you
later find a contradiction, try one of the other orders.

4b. Determine whether a DCO with your arrangement will produce the
observed DCO phenotypes.
– You will encounter a contradiction unless you have chosen the correct
gene order. Keep trying until you get the right one.
 To calculate recombination %:
• Total crossovers between y and w (SCO1 + DCO) :

(80  70  3  3)
  1.56%
•
1000
Total crossovers between w and ec (SCO2 + DCO) :

(193  207  3  3)
  4.06%
1000
Types of Double Exchanges:
Not All are Detectable
Genetic Map of Drosophila melanogaster
Creighton and McClintock Experiment Proved
Crossing Over was a Physical Event
• In maize, colorless (c)/colored (C), starchy (Wx)/waxy (wx) linked on
chromosome 9.

• Cytological markers on one parental homolog (knob on one end and

translocated segment on the other end) allowed direct observation.
Crossovers Between Sister
Chromatids (SCEs)
• Revealed by “Harlequin” chromosomes
labeled during DNA replication
• Occurs between mitotic sister
chromatids.
• No recombination
• Significance unknown, but increased
incidence correlated with some human
diseases.
 Bateson and Punnett Discovered Two
Traits That Did Not Assort Independently
• They suggested that the transmission of the two
traits from the parents was somehow coupled
– The two traits are not easily assorted in an independent
manner

• However, they did not realize that the coupling is

due to the linkage of the two genes on the same
chromosome

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 Morgan Provided Evidence for the
Linkage of Several X-linked Genes
• The first direct evidence of linkage came from
studies of Thomas Hunt Morgan
• Morgan investigated several traits that followed an
X-linked pattern of inheritance
• Figure 5.3 illustrates an experiment involving three
traits
– Body color
– Eye color
– Wing length

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Figure 5.3

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 P Males

P Females

• Morgan observed a much higher proportion of the

combinations of traits found in the parental generation
 Morgan’s explanation:
 All three genes are located on the X chromosome

 Therefore, they tend to be transmitted together as a unit

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 Morgan Provided Evidence for the
Linkage of Several X-linked Genes
• However, Morgan still had to interpret two
key observations

– 1. Why did the F2 generation have a significant

number of nonparental combinations?

– 2. Why was there a quantitative difference

between the various nonparental combinations?

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 Let’s reorganize Morgan’s data by considering the pairs
of genes separately

Gray body, red eyes 1,159

Yellow body, white eyes 1,017
Gray body, white eyes 17
Yellow body, red eyes 12
But this nonparental
Total 2,205 combination was rare

Red eyes, normal wings 770

White eyes, miniature wings 716 It was fairly common
to get this nonparental
Red eyes, miniature wings 401 combination
White eyes, normal wings 318
Total 2,205
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• To explain these data, Morgan considered the
previous studies of the cytologist F.A Janssens

• Janssens had observed chiasmata microscopically

– And proposed that crossing over involves a physical
exchange between homologous chromosomes

• Morgan shrewdly realized that crossing over

between homologous X chromosomes was
consistent with his data

• Crossing over did not occur between the X and Y

chromosome
– The three genes were not found on the Y chromosome

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• Morgan made three important hypotheses to
explain his results
– 1. The genes for body color, eye color and wing length
are all located on the X-chromosome
• They tend to be inherited together
– 2. Due to crossing over, the homologous X
chromosomes (in the female) can exchange pieces of
chromosomes
• This created new combination of alleles
– 3. The likelihood of crossing over depends on the
distance between the two genes
• Crossing over is more likely to occur between two genes that
are far apart from each other

• Figure 5.4 illustrates how crossing over provides

an explanation for Morgan’s trihybrid cross
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 Figure 5.4

These parental phenotypes are

the most common offspring

These recombinant offspring

are not uncommon

because the genes are far apart 5-18

 Figure 5.4

These recombinant offspring

are fairly uncommon

because the genes are very close together

These recombinant offspring

are very unlikely
1 out of 2,205

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Which possibility gives the observed results?
 Chi Square Analysis

• This method is frequently used to determine if the

outcome of a dihybrid cross is consistent with
linkage or independent assortment

• Let’s consider the data concerning body color and

eye color

• An example of a chi square approach to determine

linkage is shown next
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 Step 1: Propose a hypothesis.
 The genes for eye color and body color are

X-linked and, somehow, independently assorting

 Even though the observed data appear inconsistent with this
hypothesis, the hypothesis allow us to calculate expected values
 Indeed, we actually anticipate that the chi square analysis will
allow us to reject this hypothesis in favor of a linkage hypothesis

 Step 2: Based on the hypothesis, calculate the

expected values of each of the four phenotypes.
 An independent assortment hypothesis predicts

that each phenotype has an equal probability of

occurring
 Refer to Punnett square on the next slide
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  Total offspring equals 2,205

 Therefore, the expected

number of each phenotype is
1/4 X 2,205 = 551

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 Step 3: Apply the chi square formula

(O1 – E1)2 (O2 – E2)2 (O3 – E3)2 (O4 – E4)2

c2 = + + +
E1 E2 E3 E4

(1159 – 551)2 (17 – 551)2 (12 – 551)2 (1017 – 551)2

c2 = + + +
551 551 551 551

c2 = 670.9 + 517.5 + 527.3 + 394.1

c2 = 2,109.8

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 Step 4: Interpret the calculated chi square value
 This is done with a chi square table as shown in Chapter 2

 There are four experimental categories (n = 4)

 Therefore, the degrees of freedom (df) is n -1 = 3

 The calculated chi square value is enormous

 Thus, the deviation between observed and expected values is very
large

 According to Table 2.1, such a large deviation is expected

to occur by chance alone less than 1% of time
 Therefore, we reject the hypothesis that the two genes assort
independently
 In other words, we conclude that the genes are linked

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