LECTURE 21:

Linkage & Chi-square test

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 Linkage & Chi-squared Test Outline and
Objective
Accounting for disruptions in expected Mendelian ratios
Application of the Chi-square test

Objective: At the end of this lesson you should be able

to:
1. Define the term linked gene
2. Differentiate between results obtained from linked
genes and non-linked genes in dihybrid crosses
3. Write a null hypothesis
4. Apply the chi-squared test to results observed from
2 crosses
 LECTURE 22:
Linkage & Chi-square test
Review
We have been looking so far at inheritance of
genes on different chromosomes.
Since humans for example, have thousands of
traits, each chromosome must carry a large
number of genes.
There are only 46 chromosomes that must
carry genes of all traits.
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 Introduction - Linkage

Genes that are situated on the same

chromosome are linked and belong to the
same linkage group.
Genes in linkage groups do not normally show
independent assortment (don’t follow Mendel’s
second law).
They normally remain together and are passed
into the gametes together, and passed to the
offspring together.
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Linked Genes
Consider alleles Aa and Bb
1. If the alleles A and B are not linked (on separate
chromosomes) the gametes produced are
Parental cross Aa Bb
Gametes A a B b
F1 Genotypes AB Ab aB ab
2. If the alleles A and B are totally linked (on the same
chromosomes) the gametes produced are
Parental cross AABB aabb
Gametes AB ab
F1 Genotype AaBb
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 Linkage
Linkage reduces the number of
phenotypic classes resulting from a
cross

Using tomatoes as an example:

It is known that genes for flower colour and fruit
colour are on the same chromosome.

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 Linkage
Tomato plants with yellow flowers bear red fruit and
plants with white flowers bear yellow fruit.
W = yellow flowers; w = white flowers;
R = red fruit; r = yellow fruit.

W______R and w______r

(symbolizes genes on the same chromosome so yellow
flowers linked to red fruit and white flowers linked to
yellow fruit)

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Linkage
Linkage Parental Cross
Parents W______R x w______r
W______R w______r
Parental yellow flowers, x white flowers,
Phenotype red fruit yellow fruit
Gametes W______R x w______r
F1 Genotype W______R
w______r
F1 Phenotype All yellow flowers, red fruit

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F1 cross in totally linked genes
Linkage F1 Cross
F1 Genotype W______R x W______R
w______r w______r
F1 Phenotype yellow flowers, x yellow flowers,
red fruit red fruit
Gametes W__R w__r x W__R w__r
F2 Genotype W__R W__R w__r w__r
W__R w__r W__R w__r
F2 Phenotype yellow flowers, red fruit white
flowers,
yellow fruit
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 Linkage

If the F1 is intercrossed, and there is

total/complete linkage, two phenotypes
will be produced in 3:1 ratio (instead of
four phenotypes).

In other words, the linked dihybrid

cross behaves as a monohybrid cross.

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 Linkage

Linked genes (genes on the same

chromosome) can, however, be separated
in prophase 1 of meiosis by crossing over,
especially if they are relatively far from
each other.

So, totally linked genes are rare.

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 Meiosis I; Prophase I
Parental
Chromatids
Homologous
Chromosomes

Chromatid

Chiasma Recombinant
Maternal Paternal
Chromosome Chromosome Chromatids

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Genes on
paternal and
maternal
chromosomes
parental types
recombinant types
Homologous
Chromosomes
crossing over

13 1 from http://web.mit.edu/esgbio/www/mg/meiosis.html
 Linkage
Consider a F1 test cross (F1 x recessive).
Expected dihybrid test cross ratio is 1:1:1:1.
With linked genes, however, the following results
are obtained for the F2.
Phenotype # offspring
Yellow flowers, red fruit 68
Yellow flowers, yellow fruit 07
White flowers, red fruit 07
White flowers, yellow fruit 18

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 Linked genes
Note
The parental types are produced
Two new phenotypes called recombinants
appear.
The parental types are produced in greater
numbers than the recombinant types.
These results are explained by crossing over
which occurs in prophase 1 of meiosis when
the gametes are being formed

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 Linkage
The closer the genes are, the less likely they
will cross over.
The distance between two genes can be
determined if crossing over will separate them.
The number of recombinants in a group of
offspring can be used to calculate the crossover
value (COV).
A COV of 1% represents a distance of one unit
on the chromosome.

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 Linkage - Cross over value

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 Crossover value
A COV of 1% represents a distance of
one unit on the chromosome.
The COV can therefore be used to
determine the position of genes on
chromosomes,
The order of genes on a chromosome
can therefore be worked out
(chromosome mapping).

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 Gene
Gene pairs COV (%)
L–N 10.7
Mapping
L–X 18.5
N–X 7.8
18.5
L N X

10.7 7.8
If the distance between gene L and N is known to be 10.7 COV or units on the
chromosome.
And, the distance between L and X known to be 18.5 units.
If N is between L and X, the distance between N and X can be calculated to be
7.8 units.
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22
 Chi-square (X2) test

The X2 test is a way of estimating the probability

that differences between observed and expected
results are due to chance.
used to determine if the number of progeny of a particular phenotype
observed is due to linkage or chance.

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 Chi-square (X2) test
The following example is from Applications of Genetics (Jennifer
Gregory, 1996), Cambridge University Press. Page 12.

Pure breeding Drosophila with straight wings

and grey bodies were crossed with pure
breeding curled-winged and ebony bodied flies.
The F1 were all straight winged, grey bodied.

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 Linked genes
The female flies from F1 were test crossed with the
curled–winged ebony-bodied males.
The observed results follow:

(straight-wing, grey-body x curl–wing, ebony-body)

Straight wing, grey body 113
Straight wing, ebony body 30
Curled wing, grey body 29
Curled wing, ebony body 115
o Total Progeny 287

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 Linked genes
F1 test cross; observed vs. expected

The observed ratio is approximately 3:1:1:3

This is a dihybrid test cross therefore the
expected is ratio is 1:1:1:1
You need to know expected ratios!
The expected values are (1/4 x 287) = 71.5
progeny of each phenotype

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 Chi-Squared test
A chi-squared test is done to determine if the
difference between the results observed and
those that were expected is due to chance or if
they have some significance.

The null hypotheses states that the

differences between the observed and
expected values is due to chance.

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 Chi-squared test
A chi-squared table is prepared.
Calculated chi-squared = the sum of [(the observed
values - the expected values) squared, divided by
the expected values]

X 2=

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 Chi-Squared calculation

Phenotype Observed Expected O-E (O-E)2 (O-E)2/E

Values (O) Values (E)
Straight-wing, 113 71.5 + 41.25 1701.6 23.72
grey-body
Straight-wing, 30 71.5 - 41.75 1743.1 24.29
ebony body
Curled-wing, 29 71.5 - 42.75 1827.6 25.47
grey-body
Curled-wing, 115 71.5 + 43.25 1870.6 26.07
ebony-body

∑ 99.55

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 Chi-squared test
Calculated Chi-squared value =

This is to be compared with the tabulated chi-

squared value which is read off from the Chi-
squared table
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 Chi-squared test
To decide if you accept or reject the null
hypothesis (there is no difference between the
observed and accepted values) you need:
1. the calculated Chi-square value
2. the number of degrees of freedom
3. the critical value.
4. Chi-squared table of values
2, 3 and 4 are used to obtain the tabulated Chi-
square value which is compared with 1 (the
calculated Chi-squared value).
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 Chi-squared Test
Degrees of freedom (df) = n-1 where n is the
number of classes of data.
n=4 since we have 4 phenotypes
df = (4 – 1) = 3.

Critical value - by statistical convention, we use

the 0.05 probability level as our critical value.
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 Chi-squared
Procedure
Enter the chi- A Chi-Square Table
square table at df
=3 Probability
Degrees of
Look for the Freedom
0.9 0.5 0.1 0.05 0.01

corresponding 1 0.02 0.46 2.71 3.84 6.64

chi-square value 2 0.21 1.39 4.61 5.99 9.21
3 0.58 2.37 6.25 7.82 11.35
at 0.05 4 1.06 3.36 7.78 9.49 13.28
probability. 5 1.61 4.35 9.24 11.07 15.09
Copyright © 2000. Phillip McClean from
http://www.ndsu.nodak.edu/instruct/mcclean/pl
sc431/mendel/mendel4.htm

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 Chi-squared
Interpretation
If the calculated chi-square value is less than
the tabulated value at 0.05 probability, then
we accept the hypothesis that the data fits
the 1:1:1:1 ratio.

Any differences were see between the

observed and expected values are due to
chance; they are not significant.

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 Interpretation of chi-squared
If the calculated chi-square value is greater
than the tabulated value at 0.05 probability,
we reject the hypothesis that the data fits the
1:1:1:1 ratio.
This indicates that the difference between
the observes and expected values is not due
to chance; the differences are significant.
The differences are due to linkage and
crossing over.

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 Chi-squared
For the tomato example:
 Calculated chi-squared = 99.55
 Degrees of freedom = 3
 Critical value = 0.05% probability
 Tabulated chi-squared = 7.82

Inference
The calculated chi-squared is greater than the
tabulated chi-squared so the differences between the
observed and expected values are not due to chance.
They are significant (due to linked genes).

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 Example 2
In fruit flies, a certain recessive mutant allele
leads to purple eyes as opposed to the normal
red eye color.
A second allele at a separate locus leads to
vestigial wings as opposed to normal length
wings.
A test cross was conducted for these two loci
by the biologist J.B. Morgan in the early part of
the 20th century. This test cross took F1 females
from a standard dihybrid cross and crossed them
with a male pure breeding for purple eyes and
vestigial wings (homozygous recessive).
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 Example 2
Results observed
Progeny phenotype # of progeny
red eye, normal wing 1139
purple eye, vestigial wing 1195
red eye, vestigial wing 152
purple eye, normal wing 154

Total Progeny 2640

Expected # of each type of progeny = ¼ x 2640= 660
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Chi-squared Calculation
Phenotype Observed Expected O-E (O-E)2 (O-E)2/E
Values Values (E)
(O)
red eye, normal 1139 660 479 229441 347.4
wing

purple eye, 1195 660 535 286225 433.7

vestigial wing

red eye, 152 660 -508 258064 391.0

vestigial wing

purple eye, 154 660 -506 256036 387.9

normal wing

∑ 1560

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 Tabulated chi-squared value
Probability
Calculated chi-
Degrees of
squared value =1560 Freedom
0.9 0.5 0.1 0.05 0.01
DF = 3 1 0.02 0.46 2.71 3.84 6.64
0.05% probability 2 0.21 1.39 4.61 5.99 9.21
Tabulated chi- 3 0.58 2.37 6.25 7.82 11.35
squared value =7.82 4 1.06 3.36 7.78 9.49 13.28
5 1.61 4.35 9.24 11.07 15.09

Results: calculated chi-squared is greater than the

tabulated chi-squared so the result differences
between the observed and expected values are
significant.
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Example 3
Phenotype O E O-E (O-E)2 (O-E)2/E
red eye, 183 164 18.8 351.56 2.14041096
normal wing
purple eye, 165 164 0.75 0.5625 0.00342466
vestigial wing
red eye, 152 164 -12 150.06 0.91362253
vestigial wing
purple eye, 157 164 -7.3 52.563 0.32001522
normal wing
657 3.37747336

Results: calculated chi-squared (3.4) is less than

the tabulated chi-squared (7.8) so the result
differences between the observed and expected
values are not significant. The genes are not linked.
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 Gene interaction

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 Multiple alleles (multiple
allelomorph)
This is a condition where a single gene has more than
2 possible alleles
Example: blood group; this is one gene with three
possible alleles A, B,O and Coat and eye color in mice

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 Epistasis
Arises when the alleles of one gene suppresses or
masks the action of another
Example occur in mice where three genes determined
coat color.
The absence however of a dominant allele at one of the
loci results in no pigment being produced and the coat
being albino
This occurs regardless of the genes present at the other
loci , even if these produce normal coat color,
The gene at the third locus suppresses the action of the
others

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 Polygenes
Many genes acting together to cause a single
phenotype are referred to as polygenes
Polygenes gives rise to continuous variation

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 Codominance
This is where two or more alleles do not show
complete dominance or recessiveness due to failure of
any allele to be dominant in the heterozygous
condition
Found in both plants and animals
The heterozygote has an intermediate phenotype where
both alleles are expressed
Example: AB blood type

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47
 Variation

Continuous and Discontinuous

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 Variation
Describes the difference in the characteristics shown
by organisms belonging to the same natural population
or species
Based on a study on phenotypic differences two form
of variation exist
Continuous
Discontinuous

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 Discontinuous ( discrete variation)
Exhibits a limited form of variation.
Produces individuals showing clear cut differences with no
intermediates between them
The frequency distribution of this type of variation can be
shown using a bar graph
Since the phenotypic variation is restricted to certain clear cut
characteristics this form of variation is alternatively known as
qualitative inheritance as oppose to quantitative inheritance
which is a characteristics of continuous variation
Usually controlled by on or two major genes having two or
more allelic forms
Phenotype unaffected by environmental conditions

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 Continuous variation
Shows a complete graduation from one extreme to the
other without any break
The frequency distribution for continuous variation is
shown using a distribution curve/ gaussian curve or a
histogram
Caused by the combined effect of many genes
(polygenes) and environmental factors
Influenced heavily on environmental factors

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 Question
What are some sources of variation ?

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 Sources of variation
Crossing over
Independent assortment
Random fusion of gametes

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 Mutation

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 Mutation
Any change in the structure or the amount of DNA of an
organism is called a mutation.
Most mutation occur in somatic (body) cells and are not
passed from one generation toto the next.
Only those mutation that occur in the formation of
gametes can be inherited
These mutations produced sudden and districted differences
between individuals. They are therefore the basis of
discontinuous variation
There are two types of mutation:
 Gene mutation and chromosomal mutation

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 Gene mutation ( point mutation)
This is a change in the DNA structure occurring at a
single locus on a chromosome
Any change in the sequence of the nucleotides of the
DNA molecule
This produces the wrong sequence of amino acid in the
protein it makes
Example of gene mutation could be the absence of a
pigment such as what causes albinism
Other example: sickle cell anemia, cystic fibrosis

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 Forms of gene mutation
Duplication: a portion of the nucleotide chain becomes
repeated
Addition ( insertion): an extra nucleotide sequence becomes
inserted in the chain
Deletion: a portion of the nucleotide chain is removed from
the sequence
Inversion: a nucleotide sequence becomes separated from the
chain, it rejoins in its original position, only inverted . The
nucleotide sequence of this portion is therefore reversed
Substitution: on of the nucleotides is replaced by another
which has a different organic base

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 Chromosomal mutation
Chromosomal mutation can be place in three types
Whole set mutation
Chromosomal number mutation
Chromosomal structure mutation

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 Whole Set mutation

This is when organisms have an additional whole set

of chromosome
 Instead of having haploid sets in sex cells and diploid sets in
body cells they have several complete sets
 This is know as polyploidy
Know what is triploid and tetrapliod

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 Chromosomal number mutation
Caused by an individual chromosome failing to
separate at anaphase
This is cause by non-disjunction where the chromosome
fail to separate at anaphase resulting in one cell getting
too much chromosomes and another getting too little
Example of this is down syndrome (mongolism)

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 Down’s syndrome
In this case the 21st chromosome fail to separate and
the gametes produced contain 24 chromosomes
The fusion of this cell with another cell with 23
chromosomes will result in 47 chromosomes
Down’s syndrome children have varying disabilities
Typically they have a flat, broad face, squint eyes with
skin fold in the inner corner and a furrow and protruding
tongue. They have a low IQ and short life span

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62
 Non-disjunction occur in the ova and not in the sperm
and is relative to age of mother
At age 45 and above the risk is increase three times
greater

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 Klinefelter’s syndrome
This is when non-disjunction occur with the sex
chromosomes.
Results in the individuals having genetic constitutions
XXY, XXXY or XXXXY
This individuals are phenotypically males but have
small testes and no sperm. There may be abnormal
breast development and body portions are generally
female

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65
 Chromosomal structure mutation
This occurs when a mistake occurs during genetic
cross over at prophase I
Read on the 4 types of chromosomal structure mutations
that can occur

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 Causing of mutation
Occurs continually
Organisms with shorter life cycles and therefore more
frequent meiotic cycles show greater rates of mutation
The rate of mutation can be increased by artificially by
certain chemical and energy sources
Any source which induces mutation is called a
mutagen
Example: high energy radiation , high energy particles
such as alpha and gamma, neutrons, nitrous acid,
mustard gas, formaldehyde

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68
 Read on genetic screening and
counseling

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