Gene Linkage

Lecture No. 1020

Petr Heneberg, E-mail: petr.heneberg@lf3.cuni.cz

 Why are the genes linked?

- There are more genes than chromosomes

- Humans have only 23 pairs of chromosomes

… but ~19,000 protein-coding genes (and thousands of genes with other functions)
= 1000x more genes than chromosomes
Tosh et al., Mammal. Genome 2022, 33: 157-168
 Why are the genes linked?
- when the chromosomes migrate to daughter cells during meiosis, they must carry more
than one gene. Therefore, these jointly carried genes are linked

- For example, human chromosome 1 has over 3,000 genes, chromosome 21 has over 400
genes, chromosome X has over 1,400 genes, and chromosome Y has over 200 genes
D. Berry, https://www.youtube.com/watch?v=84jlwjvrJwY
 Gene linkage and Mendel law

Contrary to the rule of independent assortment,

linked genes are inherited more often in identical
combinations, in which they were present already
in parental genotypes.

Deviation from Mendel‘s inheritance laws.

Mendel law of independent assortment: Genes for

different traits segregate independently during the
formation of gametes. This applies to traits that are
not located to the same chromosomes.
 Haplotype

Number of possible types of gametes is equal to the number of possible combinations of tracked
loci.
The alleles of genes (allelic pairs, loci), which are located on the same chromosome, tend to be
inherited together.
Haplotype is a combination of alleles that are inherited together.
Haplotype = a group of genes (loci) that are subject to gene linkage and their allels are therefore
preferentially inherited from parents to their offspring together. For example, the alleles of the
major histocompatibility complex (MHC) are inherited by means of this mechanism.
 Haplotype
• Haplotype (from Greek haploos =
simple)
• The combination of alleles that is
inherited together
• Set of two or more sites (= alleles)
of size up to the complete
chromosome; the size of the
haplotype depends on the number
of recombinations that occurred
within the set.
• Haplotype = truncated term
HAPLOid genoTYPE
• Human = diploid individual – every
human has two haplotypes of any
autosomal region Y chromosome
• A whole single haplotype is always
passed to the gamete
 Haplotype example: Y chromosome
• Men are hemizygous for characters encoded on non-homologous
parts of the Y chromosome
• Only one allele for the non-homologous part of the Y chromosome
• The Y-haplotype transfers from father to son in practically identical
form; used in genealogical studies that look for SNP (single
nucleotide polymorphisms) or STR (short tandem repeats)
 Haplotype example: MHC complex at chromosome 6
• 6p21.3
• The genotype of the MHC chromosomal region consists of two haplotypes
• Contains three classes of genes (I, II, III), they are codominant
• One of the most polymorphic loci in the human genome (5,500 alleles)
• Glycoprotein complex located on the outer part of cytoplasmic membrane
of cells of the vertebrates
• recognizes non-self
structures

http://www.medscape.com/viewarticle/564081_5
 Dihybrid cross – incomplete linkage
Alelic pairs R and Su are localized at the same chromosome
Crossing-over may happen between them
 Gene linkage vs. independent assortment

Incomplete linkage

Genes (allelic pairs, loci), which are present

on the same chromosome are in mutual gene linkage
 Dihybrid cross according to Mendel
- Independent assortment
- Genes A and B are located at different pairs of homologous chromosomes

P: AABB x aabb (could also be AAbb x aaBB)

A A a a

B B b b

F1: AaBb

A a

B b
 Dihybrid cross according to Mendel
- Independent assortment
- Genes A and B are located at different pairs of homologous chromosomes

Gametes produced by F1: AB Ab aB ab

Ratio : 1 : 1 : 1 : 1
A A a a

B b B b

F2: Genotypes of gametes AB Ab aB ab

Ratio of gametes 1 1 1 1
AB 1 AABB AABb AaBB AaBb
Ab 1 AABb AAbb AaBb Aabb
aB 1 AaBB AaBb aaBB aaBb
ab 1 AaBb Aabb aaBb aabb

Phenotypic ratio 9 : 3 : 3 : 1
 Dihybrid cross according to Mendel - proof

Analytic backcross of the dihybrid (B1):

Also termed test backcross

Heterozygote x recessive homozygote: AaBb x aabb

P: AABB, aabb
F1: AaBb
B1: F1 x recessive homozygote = AaBb x aabb

Genotes AB Ab aB ab
B1: of gametes
ab AaBb Aabb aaBb aabb

B1 phenotypic ratio: 1 : 1 : 1 : 1

Ratios of F1 gametic genotypes a B1 phenotypes are identical

only when the independent assortment applies
The code for independent assortment is clear: B1=1:1:1:1

… but the situation is more complicated due to gene linkage.

 Who is to blame?
Gregor Johann Mendel
(1822 - 1884), augustinian clerk,
based in the city of Brno (now Czechia),
founder of genetics, established many
of the rules of heredity (Mendel laws)

Thomas Hunt Morgan

(1866 - 1945) American geneticists, naturalist and zoologist,
He studied genetic characteristics of the fruit fly (Drosophila),
Nobel prize (1933) for elucidating the role of chromosomes.

William Bateson (1861 – 1926)

British geneticist, invented the term genetics.
Popularised of the ideas of Gregor Mendel, which were
re-discovered in 1900 by de Vries and Correns.
First: Genes are arranged Morgan laws
on chromosomes in linear order.

Second: The genes of one

chromosome form a linkage
group. The number of linkage
groups of the organism
is identical to the number
of pairs of its homologous
chromosomes.

Third: Allelic exchange can occur

between the alleles
of a homologous pair
of chromosomes using crossing-
over. The crossing-over
frequency is directly
proportional to the distance
of genes on the genetic map.
Morgan`s fly room
Non-Mendelian dihybrid cross
at complete gene linkage

• The number of genes of individual organisms is always much

higher than the number of their chromosomes.
Therefore, all genes cannot be freely combinable.

• Genes that are present on one chromosome (i.e., those pairs of

alleles that are carried by one pair of homologous chromosomes)
are linked and their set forms a linkage group.

• When complete linkage applies, the examined genes A and B are

on one chromosome, and very close to one another, so there does
not occur any crossing over between them. For genes in complete
linkage, only combinations of alleles that are found in parents are
found in the offspring = haplotypes.
 Dihybrid cross at complete gene linkage

Genes A and B are very close to one another, at the same chromosome
Phase: Cis - coupling Trans - repulsion
P: AABB x aabb AAbb x aaBB
AB ab Ab aB
x x
AB ab Ab aB

A a a a
A A A a
B B b b b b B B

F1: AaBb

A a A a
B b b B
AB Ab
ab aB
 Dihybrid cross at complete gene linkage
Genes A and B are very close to one another, at one chromosome
Cis - coupling Trans - repulsion
Gametes F1: AB ab Ab aB
Ratio: 1 : 1 1 : 1
A
A

F2: AB ab Ab aB

AB AB ab Ab Ab aB
AB AB Ab Ab
ab AB ab aB Ab aB
ab ab aB aB

Genotypic ratio: 1:2:1 1:2:1

Phenotypic ratio: 3:1 1:2:1
 Dihybrid cross at complete gene linkage
Analytic (test) backcrossing of the dihybrid – B1:
Heterozygote x recessive homozygote: AaBb x aabb
cis trans
Gametes F1: AB ab Ab aB

A a A a

B b b B
B1: a AB ab Ab aB

ab AB ab ab Ab aB
b ab ab ab ab

Phenotypic ratio: 1 : 1 1 : 1

• The F1 dihybrid forms ONLY two types of gametes, both with

non-recombined (parental) combinations of alleles, in a ratio 1:1
 Dihybrid cross – incomplete linkage
Allelic pairs A and B are localized at the same chromosome
Crossing-over may happen between them

• Gametes with a set of alleles that are in gene linkage, and

which is different from the parental set, are created when
there have occurred a recombination between the alleles

• The mechanism of recombination is termed crossing-over

• The closer the locuses are to one another on the chromosome,

the less likely it is that there will be a simple cross-over

• The crossing-over frequency is directly proportional to the

distance of the genes on the genetic map
Crossing-over and recombination (during meiosis)
 Dihybrid cross – incomplete linkage
Allelic pairs A and B are localized at the same chromosome
Crossing-over may happen between them

Phase: Cis - coupling Trans - repulsion

AB ab Ab aB
P: x x
AB ab Ab aB

A A a a A A a a

B B b b b b B B

F1: AB Ab
ab aB
A a A a

B b b B
 Dihybrid cross – incomplete linkage
Allelic pairs A and B are localized at the same chromosome
Crossing-over may happen between them

Phase: Cis - coupling Trans - repulsion

Gametes F1:
Ratio: AB > Ab aB < ab AB < Ab aB >ab
Parental (non-recombined) AB ab Ab aB
Recombined Ab aB AB ab

A a A a A a a A
> >
B b b B b B b B
 Dihybrid cross – incomplete linkage
Allelic pairs A and B are localized at the same chromosome
Crossing-over may happen between them
Genotypes of gametes AB Ab aB ab
Ratio 2 1 1 2
AB 2 4 2 2 4
F2 (cis):
Ab 1 2 1 1 2
aB 1 2 1 1 2
ab 2 4 2 2 4
Phenotypic ratio: 22 : 5 : 5 : 4

Genotypes of gametes AB Ab aB ab
F2 (trans): Ratio 1 2 2 1
AB 1 1 2 2 1
Ab 2 2 4 4 2
aB 2 2 4 4 2
ab 1 1 2 2 1
Phenotypic ratio: 19 : 8 : 8 : 1
 Dihybrid cross – incomplete linkage
Analytic (test) backcrossing of the dihybrid – B1:
Heterozygote x recessive homozygote: AaBb x aabb
F1 gametes: AB Ab aB ab

A A a a

B1: B b B b

a Genotypes AB Ab aB ab
of gametes
b ab AB Ab aB ab
ab ab ab ab

Phenotypic ratios: cis 2 : 1 : 1 : 2

trans 1 : 2 : 2 : 1
 Dihybrid cross – incomplete linkage
• Consequences of incomplete gene linkage in dihybridism: in the
offspring of F2 and B1 generations, there is a greater proportion of
parental allele combinations than recombinant
• In the F2 generation, there are
the same genotypes as
in dihybrids that are subject
to independent assortment
but the phenotypic ratios
are different
• In the B1 generation, the F1
parent forms the same types
of gametes as in dihybrids
that are subject to independent
assortment but the gametes
are not formed in a ratio
of 1: 1: 1: 1 as in case
of independent assortment
 How can we measure gene linkage strength?
• Genetic distance
• Based on the number of recombinant and non-recombinant
offspring in the analytical (test) backcross – B1
• The number and ratio of phenotypes in B1 show, whether the genes
are subject to independent assortment, or whether
they are completely
or incompletely linked

• Expressed with the help

of Morgan's and / or
Bateson's numbers
Genetic distance based on gene linkage

Morgan's number = p
Strength of fr
= p
gene linkage fr + fnr

fr – frequency of recombinant gametes

fnr = frequency of parental gametes
0 cM < p < 50 cM
p = 0 cM complete gene linkage

p = 50 cM independent assortment
• Gene map unit: cM = centimorgan = % of recombinants = relative
distance of alleles = genetic mapping unit
• 1cM = recombination frequency of 1% (0.01M)
Bateson's number = c
The information on how many times more frequent are the
parental combinations relative to the recombinant ones

fAB + fab
Cis phase: c =
fAb + faB

Trans phase: c = fAb + faB

fAB + fab

1 < c < ∞

c = 1 = Independent
assortment
 Gene linkage analysis = genetic mapping

• Indirect method
• We are looking for a mutated gene (locus) causing a genetic disease
• The mutant locus is tested for its gene linkage with a set of DNA markers
with known position of chromosomes
• The aim is to find the linkage between the mutated gene and a DNA marker,
then all candidate genes proximal to the merker are tested for their
variations = positional cloning of the mutation
• A simpler alternative: determining the order of the loci by a three-point
test cross, in which we simultaneously monitor gene linkage (recombination
frequencies) of 3 different genes, e.g., A, B and C.
 Three-point test cross
1) Let`s have two genes with known position, named A and C. We aim to find
the position of the gene B relative to the genes A and C.

2) To perform the three-point test, we cross the recessive homozygote

(aabbcc) with the F1 hybrid (AaBbCc).

3) Next, we assess relative abundance of genotypes of the offspring:

a) ABC/abc or abc/abc = 75 %
b) aBC/abc or Abc/abc = 15 %
c) ABc/abc or abC/abc = 9 %
d) AbC/abc or aBc/abc = 1 %

4) The least represented group (AbC or aBc) are individuals, in which two
recombinations occurred during a single meiosis, which is the least
probable option (as opposed to no recombination and a single
recombination only). Thanks to two recombinations that occurred
between the centrally located gene and the two genes at the border, the
centrally located gene can be identifed.
 Three-point test cross
- We determine the parental genotypes.
They are always the ones with the
highest frequencies. Here, the parental
genotypes are ABC and abc.
- We specify the order of the genes. We
start from the knowledge of double crossing-over, which we determine
from the lowest frequency of genotypes - here AbC and aBc.
We determine the linkage distances between the genes.
- We can see that the B gene must be in the middle because the recessive allele b is now
on the same chromosome as alleles A and C and the dominant allele B is on the same
chromosome as the recessive alleles a and c.
- In the above example, the centrally located gene is the gene B; therefore the order of
genes is A-B-C or C-B-A (only one option is correct; the choice needs to be done based
on our knowledge of the position of the two known marker genes on the chromosome).
- We determine the genetic distances between genes A-C and C-B.
- The linkage is calculated as a proportion of the total number of recombinant gametes to
the total number of gametes (Morgan's number p).
- If the calculated distance differs, double crossing-over must be taken into account.
 Factors that affect the gene linkage strength

1) Multiple crossing-over
a) Reciprocal – affects only two of the four non-sister chromatids
b) Complementary – one affects one pair of non-sister chromatids; another affects the second pair
c) Diagonal – three chromatids participate in this crossing-over; one chromatid undergoes two crossing-overs
d) Crossing over of two, three or four chromatids

2) Interference – manifestation of crossing-over at a certains site prevents the origin of

another crossing-over in its vicinity
a) If crossing over probability between A-B is p=0.2 and between B-C p=0.2, the probability of double-crossing over
equals theoretically to 0.2 x 0.2, but the frequency of such crossing-overs is even lower in the reality
b) Coincidence coefficient: ratio of observed and theoretical number of double-recombinants

3) Absolute linkage – crossing over is prevented at all or results in nonviable recombinants

4) Pseudoallelism – two genes with similar functions are located so close to one another on a
chromosome that they are genetically linked
- this situation refers to genes that originated by duplication and code for the same phenotype.
 Application of gene
linkage – identification
of causes of cancer
Gene linkage application – mapping of disease loci

Polymorphic markers:
- A marker that is frequently heterozygous in the population
- One can therefore distinguish the two copies of a gene that an individual inherits
- They are not themselves pathological - they simply mark specific points in the genome

Detection:
- Variable number of tandem repeats (VNTRs) – Changes in the numbers of repeated DNA
sequences arranged in tandem arrays

3-repeat allele

4-repeat allele

ACGTGTACTC

- Microsatellites – Particular class of VNTR with repeat units of 1-6bp in length. The most widely
used are the CAn microsatellites

6 (CA) allele CACACACACACA

8 (CA) allele CACACACACACACACA

- Single nucleotide polymorphisms (SNPs) – polymorphisms due to a base substitution or

insertion or deletion of a single base
Gene linkage application – mapping of disease loci

The genotype for a microsatellite

marker on chromosome 1

Paternal copy Maternal copy

6 (CA) allele * 8 (CA) allele *

Chromosome 1

Determine the genotype

of each family member -> Genotype of this individual in this microsatellite is (6 8)
for polymorphic markers
across the genome
Gene linkage application – mapping of disease loci
Non-informative and informative meioses

66 66 66 68 68 68 68 9 10

66 66 66 68 68 68 89 6 10

Informative
Noninformative

A lab technique used to determine whether two genetic markers are linked to one another and
how closely linked they are. It uses sexual reproduction which produces offspring in which the
two markers may have crossed over during DNA recombination.

Informative -> if repetitive sequences (markers) are different at the same location
Gene linkage application – mapping of disease loci

An autosomal dominant disease for which the

gene resides on chromosome 1

… the protocol to find this Disease-causing gene

1
Gene linkage application – mapping of disease loci
Studies that employ genetic markers

Study marker

Disease-causing gene

56 47 23 23 15 44

15 35 67 24 25 27
Gene linkage application – mapping of disease loci

Data available for genotypes of the whole family

(23) (16)

(14) (26) (34) (13) (58) (12) (13) (78) (26) (47)

(24) (46) (33) (14) (18) (25) (18) (27) (46) (67) (24)
 Využití genové vazby – mapování lokusu nemoci

Next step – to define the maximum possible extent of the region,

in which the gene linkage of markers with the study disease is manifested

Gene resides Disease-coding gene

here
Gene linkage application – mapping of disease loci
What follows? The identification of genes that are present in the narrowed interval.
Genomic databases can be used for this purpose; for example Ensembl.

www.ensembl.org
Gene linkage application – mapping of disease loci

Example: chromosome 1
 Gene linkage application – mapping of disease loci
It remains to find out the causative genetic viariation.

The identified interval of DNA is subjected to sequencing.

Monogenic diseases:
Allelic heterogeneity - different variations at the same locus (or gene) cause the same disorder.
-> β-thalassemia may be caused by several different mutations in the β-globin gene

Locus heterogeneity - Determination of the same disease or phenotype by variations at

different loci (or genes); e.g., medullary cystic kidney disease (ADMCKD)

Polygenic diseases:
Schizophrenia
Environment
Asthma
Gene 1 Essential hypertension
Phenotype Osteoarthritis
Gene 2 Type 2 diabetes
Cancer
Gene 3
-> Unrelated affected individuals
Gene 4 share ancestral risk alleles
Gene linkage application – mapping of disease loci

Polygenic phenotype

An affected individual
with unaffected parents

Affected individual joining

the family, emphasizing the
common nature of the disease

-> Unclear inheritance pattern

 Application of gene linkage
– X-linked diseases

• Linkage analysis
• Tracking common inheritance
of certain characters (diseases)
with the "marker" genes
• The father's mother genotype (grandfather) is crucial
– it provides the information about the maternal bonding phase
• In men with X-linked genes, there is no recombination,
and the mothers always inherit the only X chromosome
of her fathers
• If the offspring has X-linked markers that are not present
in the mother's father, they come from their mother (grandmothers)
• Examples: XG antigen of red blood cells, X-linked ichthyosis,
or markers of impaired color vision
 Map of a part of human X chromosome (Xq27-28)
27

17 5 12

XG Ich G6PD CV H
XG: XG antigen
Ich: X-linked ichthyosis vulgaris
G6PD: glucose-6-phosphate
dehydrogenase deficiency
CV: impaired color vision loci
H: Hemophilia
 Thank you for your attention 

× ×
 Suggested literature

• Thompson & Thompson Genetics in Medicine, 8th edition; by Nussbaum RL,

McInnes RR, Willard HF

• D. J. Pritchard, B.R. Korf: Medical genetics, Galen, 2007

• https://www.khanacademy.org/science/biology/classical-genetics/chromosomal-
basis-of-genetics/a/linkage-mapping