Chapter 9

Linkage and Genetic Mapping in

Eukaryotes and Prokaryotes
9.1 GENETIC MAPPING IN
EUKARYOTES
 LESSON LEARNING OUTCOME

1. Understand the concept of linkage and how

it relates to patterns of inheritance.
2. Understand, and be able to solve problems,
of genetic mapping in diploid plants and
animals.
3. Understand the process of genetic linkage
mapping in haploid eukaryotes.
 LINKAGE
 Eukaryotic chromosomes are long ds DNA
molecules

 Typical chromosome contains thousands of

genes (loci)

 Linkage
 loci located on the same chromosome
 linked loci tend to be transmitted as a unit
GENES LINKED ON THE SAME
CHROMOSOME SEGREGATE TOGETHER
 Each chromosome must possess
many different genes to
determine thousand of different
human characteristics.
 Any two genes which occur on
the same chromosome are said
to be LINKED!
 All the genes on a single
chromosome form a linkage
group.
 Under normal circumstances, all
linked genes remain together
during cell division and so pass
into the gamete, and hence the
offspring, together.
 They do not therefore segregate
in accordance with Mendel’s Law
of Independent Assortment.

5
 USE SYMBOL THAT SHOW THE
GENES ARE LINKED

 If the alleles A and B are not linked, diploid

genotype will be written as
AA BB or Aa Bb

 But if they are LINKED a little line is use

to symbolize the chromosome they
have in common:
AB/AB or AB/ab
6
 Crossing Over Produce Recombinant
Phenotypes

 Crossing over (meiotic recombination)

 Occurs during prophase I of meiosis at the
bivalent stage
 Non-sister chromatids of homologous
chromosomes exchange DNA segments
 Linkage Prevents
Independent Assortment

Figure 5.1
 Crossing Over May Produce
Recombinant Phenotypes

gametes with a combination

of alleles NOT found in the
original chromosomes as a
result of meiotic
recombination
These are
termed These are called nonparental
parental or recombinant gametes
gametes

Figure 5.1
AB
 Example of Linkage

 Bateson and Punnett conducted a cross in sweet

pea involving two traits
 Flower color and pollen shape

 Dihybrid cross expected to give 9:3:3:1 phenotypic

ratio of F2 phenotypes
 Observed linkage
 Called it coupling
 Example of Linkage
x

Purple flowers, Red flowers,

long pollen (PPLL) round pollen (ppll)

F1 offspring

Purple flowers,
long pollen (PpLl)

Self-fertilization

Observed Observed Expected Expected

F2 offspring phenotypes number Ratio number Ratio
(9:3:3:1)
P Purple flowers, long pollen 296 15.6 240 9
NP Purple flowers, round pollen 19 1.0 80 3
NP Red flowers, long pollen 27 1.4 80 3
P Red flowers, round pollen 85 4.5 27 1
Found F2 generation had a much greater
proportion of the two phenotypes of
parental

Discovered two traits that did not assort

independently
 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
 Body color
 Eye color
 Wing length
Linkage to a
Particular
x

Chromosome Xywm Xywm Xy+w+m+ Y

F1 generation
Sex linkage of all traits F1 generation contains wild-type
places them all on X females and yellow-bodied,
x white-eyed, miniature-winged
chromosome
males.

X y+w+ m+/ X ywm X ywm/ Y

F2 generation Females Males Total

P Gray body, red eyes, normal wings 439 319 758
Gray body, red eyes, miniature wings 208 193 401
Gray body, white eyes, normal wings 1 0 1
Gray body, white eyes, miniature wings 5 11 16
Yellow body, red eyes, normal wings 7 5 12
Yellow body, red eyes, miniature wings 0 0 0
Yellow body, white eyes, normal wings 178 139 317
P Yellow body, white eyes, miniature wings 365 335 700
 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

5-13
Reorganize Morgan’s data considering 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
 Morgan Provided Evidence for the
Linkage of Several X-linked Genes
 Morgan made three important hypotheses:

 The genes for body color, eye color and wing length are all located
on the same chromosome, namely the X chromosome. Therefore,
the alleles for all three traits are most likely to be inherited together.

 Due to crossing over, the homologous X chromosomes (in the

female) can exchange pieces of chromosomes and create new
(nonparental) combinations of alleles.

 The likelihood of crossing over depends on the distance between

two genes. If two genes are far apart from each other, crossing over
is more likely to occur between them.
 Figure 5.4

These parental phenotypes are

the most common offspring

These recombinant offspring

are common

because the genes are far apart

 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

it is product of double cross over probabilities

 GENETIC MAPPING
 Genetic mapping

 “Gene mapping”
 “Chromosome mapping”

 Determines linear order and distance between

genes linked along the same chromosome
 Each gene has its own locus at a particular site
within a chromosome
A simplified genetic linkage map of Drosophila melanogaster
GENETIC MAPPING
 Genetic maps are useful in many ways

 Allows an understanding of the overall complexity

and genetic organization of a particular species
 Comparison of genetic maps between different
species improves our understanding of their
evolutionary relationships
 Information can be used to diagnose and potentially
to treat mapped human diseases
 Helps genetic counselors predict the likelihood of
couples transmitting inherited diseases
 Can provide plant and animal breeders with
important information for selective breeding
programs
 Linkage and Genetic Maps

 Estimating the relative distances between linked

genes, based on the amount of recombination
occuring between them allows us to generate
genetic maps

 If the genes are far apart  many recombinant offspring

 If the genes are close  very few recombinant offspring
 Number of recombinant offspring X 100
Map distance =
Total number of offspring

 The units of distance are called map units (mu)

 They are also referred to as centiMorgans (cM)

 One map unit is equivalent to 1% recombination frequency

(cross over value, COV)
 Linkage Analysis and Mapping
 Genetic mapping experiments are typically
accomplished by carrying out a testcross

An individual heterozygous
for two or more genes is
crossed to an individual
homozygous recessive for
these genes

The goal: determine if recombination

has occurred during meiosis in the
heterozygous parent.
 Example of a two-point mapping cross
 Cross of two linked genes affecting bristle length
and body color in fruit flies

 e = ebony body color

 s = short bristles
 e+ = gray body color
 s+ = normal bristles

 One parent double recessive (homozygous recessive at

both loci) – s/s ; e/e
 Other parent is heterozygous at both loci (s+/s ; e+/e)
 Chromosomes are
the product of a
crossover during
meiosis in the
heterozygous parent

Recombinant
offspring are fewer
in number than
nonrecombinant
offspring
 Linkage Analysis and Mapping
 The frequency of recombination are used to estimate the
distance between the two loci

Number of recombinant offspring X 100

 Map distance =
Total number of offspring

76 + 75
= X 100
542 + 537 + 76 + 75

= 12.3 map units @ 12.3% COV

 Therefore, the s and e genes are 12.3 map units apart

from each other along the same chromosome

s e
12.3
 GENE MAPPING
CROSSOVER FREQUENCY

 In 1917, Alfred H. Sturtevant, reasoned that

different recombination frequencies
reflect different distances between genes
on a chromosome.
 The major significance of calculating
crossover frequencies is that it enables
geneticist to produce maps showing the
relative positions of genes on
chromosomes.
 For example: COV of A and B is 4%. This
means that A and B are 4 units apart on
their chromosome.

A B
4 mu 31
TAKE FIVE
 Trihybrid Crosses
 Data from trihybrid crosses can also yield information
about map distance and gene order
 The following experiment outlines a common strategy for
using trihybrid crosses to map genes
 In this example, we will consider fruit flies that differ in

body color, eye color and wing shape

 b = black body color
 b+ = gray body color

 pr = purple eye color

 pr+ = red eye color

 vg = vestigial wings
 vg+ = long wings
  Step 1: Cross two true-breeding strains that differ
at three loci.

Female is mutant Male is homozygous

for all three traits wildtype for all three
traits

 The goal in this step is to obtain F1 individuals that are

heterozygous for all three genes
 Step 2: Perform a testcross by mating F1 female
heterozygotes to homozygous recessive, male flies

 During gametogenesis in the heterozygous female F1 flies,

crossovers may produce new combinations of the 3 alleles
 Step 3: Collect data for the F2 generation
 Analysis of the F2 generation
flies will allow us to map the
three genes

 Eight possible phenotypes:

2 parental, 6 recombinant

 In the offspring of crosses

involving linked genes,
 Parental phenotypes

occur most frequently

 Double crossover

phenotypes occur least

frequently
 Single crossover

phenotypes occur with

“intermediate”
frequency
 Analysis of the F2 generation flies will allow us to
map the three genes
 The three genes exist as two alleles each
 Therefore, there are 23 = 8 possible combinations of
offspring
 If the genes assorted independently, all eight combinations
would occur in equal proportions
 It is obvious that they are far from equal

 In the offspring of crosses involving linked genes,

 Parental phenotypes occur most frequently
 Double crossover phenotypes occur least frequently
 Single crossover phenotypes occur with “intermediate”
frequency
 Step 4: Analyze data of the F2 generation

 Trihybrid testcrosses yield eight possible phenotypes in

the F2 generation
 2 parental

 6 recombinant

 2 from a single crossover

 2 from another single crossover

 2 from a double crossover

39
 The combination of traits in the double crossover tells us
which gene is in the middle
 A double crossover separates the gene in the middle from

the other two genes at either end

 In the double crossover categories, the recessive purple

eye color is separated from the other two recessive alleles
 Thus, the gene for eye color lies between the genes for

body color and wing shape

Parentals Double crossover

b pr vg b pr+ vg

b+ pr+ vg+ b+ pr vg+

 Step 5: Calculate the map distance between pairs
of genes

Map distance = no. of recombinants x 100

total no. of offspring
• To calculate the distance between b and pr

Map distance = (30 + 28 + 2 + 1) / 1005 x 100

= 6.1 mu

• To calculate the distance between pr and vg

Map distance = (61 + 60 + 2 + 1)/1005 X 100

= 12.3 mu

• To calculate the distance between b and vg

The double crossover frequency needs to be multiplied

by two because both crossovers are occurring between
b and vg

Map distance = (30 + 28 + 61 + 60 + 2[2+1])/1005 X100

= 18.4 mu
 Step 6: Construct the map

 Based on the map unit calculation the body color and

wing shape genes are farthest apart
 The eye color gene is in the middle

 The data is also consistent with the map being drawn

as vg – pr – b (from left to right)

 In detailed genetic maps, the locations of genes are

mapped relative to the centromere
QUESTION

Drosophila females of wild-type appearance but heterozygous for three

autosomal genes are mated with males showing three autosomal recessive
traits: glassy eyes (g), coal-colored body (c) and striped thoraxes (t). One
thousand (1000) progeny of this cross are distributed in the following
phenotypes classes.

Determine the gene order and draw a genetic map based on this data.

g+ c+ t+ 435
g c+ t+ 21
g c t+ 27
g+ c+ t 25
g c+ t 5
g c t 459
g+ c t 22
g+ c t+ 6
JUNE 2012
Dec 2015
 GENETIC MAPPING IN HAPLOID
EUKARYOTES
 Much of our earliest understanding of
genetic recombination came from the
genetic analyses of fungi

 Fungi may be unicellular or multicellular

organisms

 They are typically haploid (1n)

 They reproduce asexually and, in many

cases, sexually

 The sac fungi (ascomycetes) have been

particularly useful to geneticists because
of their unique style of sexual reproduction
Fungi can reproduce
sexually by the fusion of
two haploid cells to form a
zygote

Zygote can proceed

through meiosis to
produce four haploid Meiosis produces
spores four haploid cells,
termed spores
This group of four spores
is called a TETRAD
These are
In some species, meiosis enclosed in a sac
is followed by a single termed an ascus
round of mitosis to
produce eight spores 
OCTAD
SEXUAL REPRODUCTION in
ASCOMYCETES

 Ascomycetes
 “Sac fungi”
 Products of a single meiotic division are
contained within a sac known as an ascus
 Each ascus contains four
(or eight) spores
 These spores are called
ascospores
 Asci can be dissected and
each haploid spore studied
 Types of Tetrads or Octads
 The arrangement of spores within an ascus
varies from species to species
 Unordered tetrads or octads
 Ascus provides enough space for the spores to
randomly mix together
 (Fungi) Saccharomyces cerevisiae, Aspergillus
nidulans
 Unicellular algae (Chlamydomonas reinhardtii)
 Ordered tetrads or octads
 Ascus is very tight, thereby preventing spores from
randomly moving around
 (Mold) Neurospora crassa
 Tight ascus
Ascus provides prevents mixing
space for spores to of spores
randomly mix
together

Mold
Yeast
Unicellular alga
 Ordered Tetrad Analysis
 Ordered tetrads or octads have the following
key feature
 The position and order of spores within the
ascus is determined by the divisions of meiosis
and mitosis

 This idea is schematically shown in next

Figure
 The example depicts ordered octad formation in
Neurospora crassa
 Spores that carry the A allele show orange
pigmentation
 Spores that carry the a (albino) allele are white
Pairs of daughter All eight cells are
cells are located arranged in a linear,
next to each other ordered fashion
 The genetic content of spores in ordered
tetrads can be determined
 This allows experimenters to map the distance
between a single gene and the centromere

 The logic of this mapping technique is based

on the following features of meiosis
 Centromeres of homologous chromosomes
separate during meiosis I
 Centromeres of sister chromatids separate
during meiosis II
COMPARISON OF THE ARRANGEMENT OF
CELLS WITHIN AN ORDERED OCTAD

NO CROSSING OVER SINGLE CROSSING OVER

PRODUCES a 4:4 PRODUCES a 2:4:2 or 2:2:2:2
ARRANGEMENT (FDS) ARRANGEMENT (SDS)

58
This 4:4 arrangement of spores within Octad contains a linear arrangement of
the ascus is termed a first-division 4 haploid cells with the A allele which
segregation (FDS) or an M1 pattern are adjacent to 4 with the a allele

Because the A and a alleles have

segregated from each other after
meiosis I

Figure 5.14 (a) No crossing over

These arrangement of
spores are termed a The A and a alleles do not
second-division segregation segregate until meiosis II
(SDS) or M2 patterns

Figure 5.14 (b) Single crossing over

 The percentage of M2 asci can be used to calculate
the map distance between the centromere and the
gene of interest
 Therefore the chances of getting a 2:2:2:2 or 2:4:2 pattern
depend on the distance between the gene of interest and the
centromere

 To calculate this distance, the experimenter must count the

number of SDS asci, as well as the total number of asci
 In SDS asci, only half of the spores are actually the

product of a crossover
 Therefore

(1/2) (Number of SDS asci) X 100

Map distance =
Total number of asci

SDS = 2ND division segregation (asci with arrangement 2:2:2:2 and 2:4:2)
 Mapping the
distance
from a gene
to the
centromere

4:4

2:4:2

2:2:2:2
63
 TUTORIAL
 In a cross in Neurospora involving two alleles, B
and b, the following tetrad patterns were
observed. Calculate the distance between the
gene and the centromere.

Tetrad pattern Number

BBbb 36
bbBB 44
BbBb 4
bBbB 6
BbbB 3
bBBb 7
QUESTION 5 – APR 2009
In a cross in Neurospora where one parent expresses the mutant
allele (a) and the other expresses a wild type phenotype (+), the
following data were obtained in the analysis of ascospores:
Asci Type
1 2 3 4 5 6
+ a a + a +
+ a a + a +
+ a + a + a
+ a + a + a
a + a + + a
a + a + + a
a + + a a +
a + + a a +
39 33 5 4 9 10

Calculate the gene to centromere distance.