LINKAGE AND

CONSTRUCTION OF GENETIC MAPS

Dr.S.N.Hegde
Professor, Department of Zoology
University of Mysore
Mysore – 570006
E-mail: malerhegde@yahoo.com
Cell: 9448365799
Map ?

Physical?
Cytological ?

Genetic ?
Linkage ?
• Physical map:

• Restriction map
 E1 E2 E3
GEN ET ICS
Processed mRNA
Or cDNA
Black bars represent
the restriction sites

GEN ET ICS
DNA
E1 I1 E2 I2 E3

•Restriction map of native DNA and that of a

processed mRNA
•Use same restriction enzyme for comparison

Restriction maps of different organisms

or genotypes are not similar
• Quantitative Trait Loci (QTL)

• Restriction Fragment Length

Polymorphism (RFLP)

P1

P2

Offspring
• Recombination within
restriction fragments
• Recombination frequency is
calculated
• Maps are constructed
SNPs and Mapping:
• SNPs (or "snips") = DNA sequence
variations that occur when a single
nucleotide (A,T,C,or G) in the genome
sequence is altered.
• Eg. DNA sequence AAGGCTAA
changed to ATGGCTAA.
• This results in DNA variation
• For a variation to be considered a SNP,
it must occur in at least 1% of the
population.
• SNPs, which make up about 90% of all
human genetic variation, occur every
100 to 300 bases along the 3-billion-
base human genome.
• Two of every three SNPs involve the
replacement of cytosine (C) with
thymine (T).
• SNPs can occur in coding (gene) and
noncoding regions of the genome.
• Many SNPs have no effect on cell
function, but scientists believe others
could predispose people to disease or
influence their response to a drug.
• Although SNPs contribute only 1% of human
DNA sequences, variations in DNA sequence
can have a major impact individuals
response to disease; environmental factors
such as bacteria, viruses, toxins, and
chemicals; and drugs and other therapies.
• This makes SNPs valuable for biomedical
research particularly personalized drug
design.
• Making population studies easier.
• Genome map is available
• SNP variants are detected and data
base is created
• The genome with a mutant allele is
amplified, sequence is read, compared
with SNP database for its map position
Cytological maps:
Polytene chromosome of Drosophila
Polytene chromosome map of
Drosophila melanogaster
Polytene chromosome arm of 3L
of Drosophila melanogaster
Construction of genetic maps using
deletions
on the polytene chromosomes
• Ring chromosomes
A B C D E F G H

3 2 1

B B
A B
A H
C C C

1 2 G
3
G D
D F
D F
E F E
E
Genetic map = Linkage map
second law of Mendel states
“when more than two
characters are involved in a
cross, different alleles of
these traits assort
independently in the F2
generation”
• They are randomly distributed to the
gametes and subsequently unite
independently in the next generation.
• Thus in the F2 generation the progeny
is obtained in 9:3:3:1 proportion.
• Test cross would give 1:1:1:1 ratio
Yellow/Round X Green/Wrinkled

yy rr
YY RR

y r Gametes
Y R

Y y R r F1 hybrid
 Yy Rr

Meiosis

Y R y R Y r y r

F1 Gametes
• Discovery of interaction of genes
• Mendelian ratios cannot be universally
applied.
• Bateson and Punnet were studying
inheritance of certain characters in
sweet peas.
• They crossed the plants bearing purple
colored flowers (P) and long pollen (L)
with those producing red flowers (p)
and round pollen.(l).
 Flower color and nature of pollen

PPLL X ppll
Purple long red round

Purple long F1 -
(P-L-)

F2 Progeny Test cross

Purple Long (P-L-) ……… 284
Purple round (P-ll) ……… 21
Red long (ppL-) ……… 21
Red round (ppll) ……… 55
-------------
Total 381
-------------
Expected: 215:71:71:24 = 381
 PPll X ppLL
Purple round red long

Purple long F1 -
(P-L-)
F2 Progeny
Test cross
Purple Long (P-L-) ……… 15
Purple round (P-ll) ……… 144
Red long (ppL-) ……… 136
Red round (ppll) ……… 19
-------------
Total 314
-------------
• In both the crosses some combinations
appeared more than expected while others
less than expected.
• Bateson and Punnet thought that these
two classes were more because of excess
of two gametic types PL and pl in the first
and Pl and pL in the second.
• Because these two types were found in
the parental gametic types, they thought
that it is due to close association or
coupling of two dominant or two
recessive genes together in the first.
• In the second set, the two dominant or the
two recessive genes tended to separate,
and this phenomenon was termed as
repulsion.
• Thomas Hunt Morgan introduced
Drosophila as a genetic tool
• The explanation of Bateson and Punnet’s
results could be given only after this.
• Morgan similarly found deviation from
Mendel’s dihybrid ratios while studying two
autosomal gene pairs in Drosophila.
• In Drosophila, the normal eye color is red
and there are number of mutations
affecting the color of eyes. Purple is a
mutant.
• Similarly there are also number of mutant
genes, which affect the shape, or nature
of wings. Vestigeal is a wing mutation
Morgans experiment
Mutant X Wild type
Purple Vestigial Red eye, normal wing
prprvgvg
pr+pr+vg+vg+

F1 Normal female X Purple Vestigial male

pr+prvg+vg prprvgvg
Test cross
pr+vg+ .… 1339
prvg …. 1195
pr+vg .… 151
prvg+ .… 154
-----------
Total 2839
-----------
• There was deviation from Mendelian
1:1:1:1 ratio and they indicate coupling.
• This shows that the gene combination in
the gametic population of one sex in F1
showing coupling.
 Purple Normal X Normal vestigeal
prprvg+vg+
pr+pr+vgvg

F1 Normal female X Purple Vestigial male

pr+prvg+vg prprvgvg

Test cross
pr+vg+ .… 157
prvg …. 146
pr+vg .… 965
prvg+ … 1067
-----------
Total 2335
• Here double dominant or double recessive
combinations appeared with low frequency
than one dominant and one recessive
character.
• This phenomenon was the repulsion of
Bateson and Punnet. The nonallelic
dominant genes repelled here.
• Morgan suggested that in coupling both
pairs of genes are located on the same
chromosome, hence they have a tendency to
move to the same gamete.
• In first set of experiment, both prvg and
pr+vg+ are located on the same
chromosome, while in the second set pr+ vg
on one homologue and pr vg+ on the other
homologue.
• This helped to explain the phenomenon of
coupling and repulsion. The first situation is
coupling and the second is repulsion.
• Morgan suggested that the genes located on
chromosomes.
• He also thought that the genes located on the
same chromosome have a tendency to move
together during gamete formation and those
located on different chromosomes assort
independently.
• This phenomenon of placement of genes on
the same chromosome was called linkage
• Homologous chromosomes pair during
meiosis, occasionally crossing over
takes place between them.
• Original gene arrangement on two
chromosomes is called parental
combination.
• New combinations are called cross
over types or recombinants.
• The evidence for this exchange comes
from the chiasma formed during
meiosis.
• The chiasma is visible expression of
genetic crossing over.
• The deviation from the Mendelian ratios
in above crosses was because the two
genes are located on the same
chromosome hence they did not assort
independently.
• Linkage is a situation in which gene pairs
reside on the same chromosome.
• The two gene pairs present on the same
chromosome are said to be linked.
• In Morgans experiment, linkage of the two
dominant alleles of the two genes together
on the same chromosome or the two
recessive genes together is coupling
• and repulsion is the linkage of one dominant
and one recessive alleles of the gene.
• Morgan and his associates found that
numerous hereditary traits are
associated with each chromosome, and
they have tendency to be inherited
together.
• Such groups were called linkage
groups.
• Each organism has large number of
hereditary characters or genes and few
chromosomes.
• If Suttons and Morgan’s views that the
genes are located on chromosomes are
correct, then it is obvious that each
chromosome should have several
genes located on it.
• Examples:
• In humans, 30,000 genes and 23
chromosomes
• In Drosophila, 5000 genes and 4
chromosomes
• All genes located on a chromosome
have tendency to be inherited together
as group.
• Each group of genes inherited together
as one unit is called a linkage group.
• The number of groups of genes will
represent the number of chromosomes
or
• The number of linkage groups is equal
to the number of chromosomes in the
haploid set.
• Examples:
Drosophila – n = 4, Linkage group 4.
X – rod like, 2nd and 3rd are Vs and 4th is
dot
First chromosome (X) – Linkage group 1,
2nd chromosome – 2nd linkage group,
3rd chromosome – 3rd linkage group
and 4th chromosome – 4th linkage group
• Human – n =23, 23rd is sex chromosome
• The whole genome of Drosophila is
analyzed now.
• The fourth chromosome has only few
genes
• Second and third contain most of the
genes.
• The number of genes present on a
linkage group depends on the physical
size of the chromosome.
Number of chromosomes and linkage
group in some organisms
Sl.N Species Haploid Linkage
o No. group
1 Maize 10 10
2 Mouse 20 20
3 Man 23 23
4 Onion 8 8
5 Pea 7 7
6 Silkworm 28 28
• In Drosophila melanogaster the yellow
and white eye genes are located on the
first linkage group.
• When a heterozygous female (y+y/w+w)
is crossed with wild male parent
produces only either wild males or
yellow-white mutant.
• Small proportion of yellow normal
(yw+) and normal white(y+w) flies will
also appear and their frequency is less.
In Drosophila yellow and white are on X chromosome

y+ y y+
X
w+
w+ w

y+ y+ y y+ y+ y

w+ w+ w+ w
w+ w
 y+ y y+
X
w+
w+ w

y+ y y+ y+ y
y+

w w+ w+ w+ w w+
Bent and shaven are on 4th chromosome

bt+
bt
X
sv+ sv

bt

sv
 bt+ bt bt
X sv
sv+ sv

bt bt
bt+ bt bt bt+
sv sv
sv+ sv sv sv+

•All crosses gave only parental combinations,

no recombinants appeared
•This is because of close association
•This is called complete linkage
• Complete linkage is not only restricted
to the IV chromosome of Drosophila.
• It is also seen for all gene pairs when
heterozygous male is used as a parent
• Morgan concluded – No crossing over
in male Drosophila
• The occurrence of recombination is
due to crossing over, which takes
place in the F1 females.
• Morgan assumed that the genes occur
on the chromosomes in a linear order
along the length of the chromosome.
• Thus linkage is the physical
relationship between genes and
chromosomes and crossing over is
the exchange of genes between
homologous chromosomes.
• Janssen had already demonstrated the
presence of chiasma cytologically and
Morgan’s work supported that of
Janssen.
• However complete linkage between
genes on the same chromosomes is
only a rarity.
• CONSTRUCTION OF LINKAGE MAPS
IN DIPLOIDS
• On the basis of recombination
frequencies, linkage maps can be
constructed.
• Eg. In Morgans experiment, out of 2839
• Pr+Vg+ = 1339 + prvg = 1195,
= Total 2534 (PC)
• Pr+vg = 151, prVg+ = 154 :
=Total 305 (RC)
• The recombination frequency for Pr+
and Vg+ is 10.74 (305X100/2839).
• Sturtevant found that the
recombination frequency were different
for different gene pairs.
• He suggested that this frequency is a
quantitative index of linear distance
between two gene pairs on a
chromosome, hence it could be used to
map the genes on the chromosome.
• The idea was simple.
• During meiosis, crossovers occur randomly
along the paired homologs.
• If crossing over occurs between chosen gene
pairs then alone recombinants appear.
• No recombinants appear if crossing over
occurs elsewhere.
• Further the proportion of crossing over is
higher if the two genes are placed apart.
• Thus by measuring the recombination
frequency distance between gene pairs can
be measured.
• One map unit is the distance between
gene pairs for which one product is a
recombinant out of one hundred
meiotic divisions.
• A recombination frequency of 0.01 or
1% is defined as one map unit, or
morgan unit.
• The place on the map that represents a
gene pair is called locus.
• The map for pr and vg may be
diagrammatically represented as follows,
pr vg
10.7

Two point cross and three point crosses

Three point cross : Bridges and Olbright
Test cross

+ sc + sc sc

X ec + ec X ec

cv + cv cv

+
+ + + = 1174 Parental combinations
sc ec cv = 934
+ ec cv = 99 Recombinations between sc – ec
239X100/2635=9.1%
sc + + = 140
+ + cv = 124 Recombinations between ec-cv
288X100/2635=10.9%
sc ec + = 164
Total recombinants :
Total = 2635 527X100/2620.035=20.0%
 Genetic map of sc-ec-cv

sc ec cv

9.1 10.9

20.0
Theoretically 8 combinations are
possible
But in the previous example only six
classes were present
When more than 8000 flies were counted
in the progeny Bridges and Olbright
obtained following results
+ + + = 8576 Parental combinations
sc ec cv = 8808
+ ec cv = 671 Recombinations between sc – ec
1387X100/20785=6.72%
sc + + = 716
+ + cv = 1002 Recombinations between ec-cv
1999X100/20785=9.62%
sc ec + = 997
sc + ec = 4 Double cross overs
5X100/20785=0.02%
+ ec + = 1

Total recombinants :
Total = 20785 527X100/2620.035=20.0%
 Genetic map of sc-ec-cv

sc ec cv

6.72+.02=6.74 9.62+0.02=9.64

16.38
• Three point cross permits identification
of the central gene.
• The double crossovers appear by
crossing over between the genes
present on the two extreme sides and
the central gene.
• Further the total recombination
frequency or the distance between the
two outside genes is the highest.
• Linkage maps of many organisms have
been constructed based on such
recombination studies.
• The place on a chromosome where a
gene is located is called locus (plural
loci).
• The alleles of the same gene whether
wild type or mutant have same location.
COINCIDENCE AND INTERFERENCE
• In the above crosses recombination
occurs at two places, i e. between sc-ec
and ec-cv.
• The double crossovers also occur
although at low frequency.
• Are these two crossovers independent or
depend on one another?
• If crossing over at one place does not
affect the crossing over at the other,
then the probability of double
crossovers could be calculated as the
product of the occurrence of the event
at each place.
• Accordingly, here, out of 20785 flies, a
proportion of 0.0674 were crossovers
between sc-ec and 0.0964 were between ec-
cv.
• The product of these two events is 0.0674 X
0.0964 = 0.00650;
• No. of flies expected = 0.00650 X 20785 = 135.
• while observed is only 5 (0.00024)flies.
• This shows that the crossing over at one
region interferes with the crossing over in
the second region. This phenomenon is
called interference.
• The occurrence of 5 double crossovers was
because of coincidence.
• Muller calculated the ratio of observed
frequency of double crossovers to the
frequency of expected double crossovers
and this value is called coincidence
coefficient.
• The coincidence coefficient in the present
experiment is 0.00024/0.00650 or 5/135 =
0.037 or 3.7%. The interference value may
calculated by deducting the coincidence
coefficient by one or 1-0.037 = 0.963 or
96.3%.
• The linkage maps are essential for
the genetic study of an organism.
• The linkage maps and knowledge of
coincidence coefficients enables to
understand expected recombinants.
• Nowadays the genes are manipulated
through genetic engineering.
• Unless the position of the genes on
the chromosome is known, it is not
possible to manipulate it.