4

Gene Linkage and

Genetic Mapping

1
If two genes are on
different chromosomes…

2
If two genes are on
different chromosomes…

Half look like they got a

set of the parents
chromosomes…

And half look like they got

a mix of both parents
chromosomes…
3
Now suppose both gene A
and B were next to each
other on the same
chromosome.

What happens to the ratios

in this diagram?

4
5
6
 Gene Mapping
• Gene mapping determines the order of genes and the relative
distances between them in map units

• 1 map unit = 1 cM (centimorgan)

• Gene mapping methods use recombination

frequencies between alleles in order to determine the relative
distances between them

• Recombination frequencies between genes are inversely

proportional to their distance apart

• Distance measurement: 1 map unit = 1 percent recombination (true

for short distances)
7
Fig. 4.6 8
 Gene Mapping
• Genes with recombination frequencies less than 50 percent are on
the same chromosome = linked)
• Linkage group = all known genes on a chromosome
• Two genes that undergo independent assortment have
recombination frequency of 50 percent and are located on
nonhomologous chromosomes or far apart on the same
chromosome = unlinked

9
 Mapping the distance between two genes
Starting with pure breeding lines,
Cross Parent 1(AA BB) with Parent 2(aa bb)

So Parental chromosomes in the F1 have to be AB and ab

Now cross (AB ab) F1 progeny with (ab ab) tester

to look for recombination on these chromosomes.
Suppose you Get……

AB ab 583 <parental

ab ab 597 <parental

Ab ab 134 <recombinant

aB ab 134 <recombinant
total= 1448

so…. 268 recombinants /1448 progeny =

0.185 recombinants/progeny=
18.5% recombinants=
18.5 mu
10
 Mapping (and ordering) three genes
Starting with pure breeding lines, Cross Parent 1(AA BB DD) with Parent 2(aa bb dd)

So you know the Parental chromosomes in the F1 have to be ABD and abc

Cross (ABD abd) F1 progeny with (abd abd) tester

Ab + aB = (45+89)+(94+40) recom
268 recom/1448 total =0.185 Suppose you Get……
A-B =18.5mu
ABD abd 580 <parental
ABd abd 3
Bd + bD = (3+40)+(5+45)
93 recom/1448 total= 0.064 abD abd 5 <parental
B-D =6.4mu abd abd 592

AbD abd 45 <recombinant

Ad + aD = (3+89)+(5+94) Abd abd 89
191 recom/1448 total= 0.132
A-D =13.2mu aBD abd 94
aBd abd 40 <recombinant
total= 1448
so the order must be A-----D---B
-13.2--6.4-
----18.5----
11
So How come 13.2 + 6.4 does not equal 18.5?

We missed the double recombinants on the first pass…

Cross (ABD abd) F1 progeny with (abd abd) tester

Ab + aB = (45+89)+(94+40) recom
268 recom/1448 total =0.185 Suppose you Get……
A-B =18.5mu
ABD abd 580 <parental
(45+89)+(94+40)+2(3+5) recom ABd abd 3
284recom/1448 total = 0.196
abD abd 5 <parental
A-B =19.6mu
abd abd 592

longer the distance, more AbD abd 45 <recombinant

potential to underestimate Abd abd 89
recomb freq.
aBD abd 94
aBd abd 40 <recombinant
total= 1448
A-----D---B
-13.2--6.4-
----18.5----
12
Chromosome interference: crossovers in one region decrease the
probability of a second crossover close by

Coefficient of coincidence = observed number of double recombinants

divided by the expected number
\ If the two crossovers were independent,
we would expect that the probability of seeing two recombination events occur would be
0.132 between A-D AND 0.064 between D-B
0.132 X 0.064 = 0.008
For every 1448 progeny, this would be (1448x0.008)=12.23 double recombinants
We actually observed only (5+3)= 8 double recombinants

So the Coefficient of coincidence = observed / expected = 8/12.23 =0.65

Interference = 1-Coefficient of coincidence

Interference = 1-Coefficient of coincidence

= 1- 0.65
= 0.35
13
Mapping Genes in Human Pedigrees
• Methods of recombinant DNA technology are used to
map human chromosomes and locate genes

• Genes can then be cloned to determine structure and

function

• Human pedigrees and DNA mapping are used to

identify dominant and recessive disease genes

• Polymorphic DNA sequences are used in human

genetic mapping. 14
 Genetic Polymorphisms
• The presence in a population of two or more relatively
common forms of a gene or a chromosome is called
polymorphism
• Changes in DNA fragment length produced by presence or
absence of the cleavage sites in DNA molecules are known
as restriction fragment length polymorphism (RFLP)
• A prevalent type of polymorphism is a single base pair
difference, simple-nucleotide polymorphism (SNP)
• A genetic polymorphism resulting from a tandemly repeated
short DNA sequence is called a simple sequence repeat
(SSR)
15
 RFLPs
• Restriction endonucleases are used to map genes as
they produce a unique set of fragments for a gene

• There are more than 200 restriction endonucleases in

use, and each recognizes a specific sequence of DNA
bases

• EcoR1 cuts double-stranded DNA at the sequence

5’-GAATTC-3’ wherever it occurs

16
Fig. 4.18 17
 RFLPs
• Differences in DNA sequence generate different recognition
sequences and DNA cleavage sites for specific restriction enzymes
• Two different genes will produce different fragment patterns when cut
with the same restriction enzyme due to differences in DNA sequence

18
Fig. 4.19
 SNPs
• Single-nucleotide polymorphisms (SNPs) are abundant in
the human genome
• Rare mutants of virtually every nucleotide can probably be
found, but rare variants are not generally useful for family
studies of heritable variation in susceptibility to disease
• For this reason, in order for a difference in nucleotide
sequence to be considered as an SNP, the less-frequent
base must have a frequency of greater than about 5% in
the human population.
• By this definition, the density of SNPs in the human
genome averages about one per 1300 bp
19
 SSRs
• A third type of DNA polymorphism results from differences
in the number of copies of a short DNA sequence that
may be repeated many times in tandem at a particular site
in a chromosome

• When a DNA molecule is cleaved with a restriction

endonuclease that cleaves at sites flanking the tandem
repeat, the size of the DNA fragment produced is
determined by the number of repeats present in the
molecule

• There is an average of one SSR per 2 kb of human DNA

20
Mapping Genes in Human Pedigrees
• One source of the utility of SNPs and SSRs in human
genetic mapping is their high density across the
genome

• Additional source of utility of SSRs in genetic mapping

is the large number of alleles that can be present in any
human population.

21
Mapping Genes in Human Pedigrees

• Human pedigrees can be analyzed for the inheritance

pattern of different alleles of a gene based on differences in
SSRs and SNPs

• Restriction enzyme cleavage of polymorphic alleles that are

different in RFLP pattern produces different size fragments
by gel electrophoresis

22
A rare recessive disease that affects people late in life
is 90% linked to the RFLP marker on the gel below.

What’s the probability that II1 is a carrier of the disease? 100%

What’s the probability that the grandchildren (row III) are carriers?

dd DD

Dd
90% 10% 90%10% 10% 90% 90%

23
A rare recessive disease that affects people late in life
is 90% linked to the RFLP marker on the gel below.

What’s the probability that II1 is a carrier of the disease? 100%

What’s the probability that the grandchildren (row III) are carriers?

dd DD

Dd
10% 90% 10% 90% 90% 10% 10%

24
 Tetrad Analysis
• In some species of fungi, each meiotic tetrad is
contained in a sac-like structure, called an
ascus

• Each product of meiosis is an ascospore, and

all of the ascospores formed from one meiotic
cell remain together in the ascus

• Several features of ascus-producing organisms

are especially useful for genetic analysis:
 They are haploid, so the genotype is
expressed directly in the phenotype
 They produce very large numbers of
progeny
 Their life cycles tend to be short

25
 Tetrad Analysis
• In tetrads when two pairs B

of alleles are segregating, B

b
three patterns of b
segregation are possible
B
• parental ditype (PD) = B
two parental genotypes b
Parental
• nonparental ditype b ditype
tetrad
(NPD) = only recombinant
combinations B
B
• tetratype (TT) = all four b
b
genotypes observed
B
B
b
Parental
b ditype 26
tetrad
 Tetrad Analysis
• When genes are unlinked, the parental ditype tetrads
and the nonparental ditype tetrads are expected in
equal frequencies: PD = NPD

• Linkage is indicated when nonparental ditype tetrads

appear with a much lower frequency than parental
ditype tetrads: PD » NPD

• Map distance between two genes that are sufficiently

close that double and higher levels of crossing-over
can be neglected, equals
1/2 x (Number TT / Total number of tetrads) x 100
27
 Neurospora: Ordered Tetrads
• Ordered asci also can be
classified as PD, NPD, or
TT with respect to two
pairs of alleles, which
makes it possible to
assess the degree of
linkage between the
genes

• The fact that the

arrangement of meiotic
products is ordered also
makes it possible to
determine the
recombination frequency
between any particular
gene and its centromere
28
 Tetrad Analysis: Ordered Tetrads

• Homologous centromeres of parental chromosomes

separate at the first meiotic division

• The centromeres of sister chromatids separate at the

second meiotic division

• When there is no crossover between the gene and

centromere, the alleles segregate in meiosis I

• A crossover between the gene and the centromere

delays segregation alleles until meiosis II
29
• The map distance
between the gene and
its centromere equals

• 1/2 x (Number of asci

with second division
segregation/ Total
number of asci) x 100

• This formula is valid

when the gene is close
enough to the
centromere and there
are no multiple
crossovers

30
 Gene Conversion
• Most asci from heterozygous Aa diploids demonstrate normal Mendelian
segregation and contain ratios of
2A : 2a in four-spored asci, or 4A : 4a in eight-spored asci

Occasionally, aberrant ratios are also found, such as

3A : 1a or 1A : 3a and 5A : 3a or 3A : 5a. The aberrant asci are said to result from
gene conversion because it appears as if one allele has “converted” the other allele
into a form like itself

• Gene conversion is frequently accompanied by recombination between genetic

markers on either side of the conversion event, even when the flanking markers are
tightly linked

• Gene conversion results from a normal DNA repair process in the cell known as
mismatch repair

• Gene conversion suggests a molecular mechanism of recombination

31
•One of two ways to resolve the resulting structure, known
as a Holliday junction, leads to recombination, the other
does not

•The breakage and rejoining is an enzymatic function

carried out by an enzyme called the Holliday junction-
resolving enzyme 32
33