GENETIC LINKAGE

• Genes on nonhomologous chromosomes assort

independently during meiosis.
• Genes on the same chromosome are said to exhibit
linkage and are called linked genes.
• Linked genes, and hence the phenotypic characters they
control, are inherited together because they are located
on the same chromosome.
• Modern understanding of genetic linkage came from
the work of Thomas Morgan. Morgan showed that two
recessive genes in Drosophila melanogaster; white eye
(w) and miniature wing (m) are X-linked.
• Morgan crossed a female with white eyes and
miniature wings (wm/wm) with a wild-type male (red
eyes and large wings) (w+m+/Y).
• The F1 males were white-eyed and had miniature wings
(genotype wm/Y) while the females were wild-type for
both the eye colour and wing size (genotype
w+m+/wm).
• The F1 flies were interbred (F1 x F1) and the resulting
F2 flies were analyzed.
• In the F2, the flies fell into four classes: (a) white eyes,
miniature wings were 359 females and 391 males, (b)
wild-type (red eyes, large wings) were 439 females and
352 males, (c) white eyes, large wings were 218
females and 237 males, (d) red eyes, miniature
wings were 235 females and 210 males.
• In the F2 the most frequent phenotypic classes in both
sexes were the grandparental phenotypes (class a and
b).
• The genotypes or phenotypes of the original parents are
referred to as parental classes.
• There were also flies with the nonparental phenotypic
combinations (class c and d). Nonparental
combinations of linked genes are called recombinants.
• To explain for the recombinants he proposed that in
meiosis, exchange of genes had occurred between the
two X chromosomes of the F1 females. Since males are
hemizygous, no such genetic exchange occurred
between X and Y chromosomes.
• The production of recombinants results from physical
exchanges between homologous chromosomes during
meiotic prophase I.
Crossing-over
• Crossing-over is the reciprocal exchange of
chromosome parts at corresponding positions along
homologous chromosomes by symmetrical breakage
and rejoining. Crossing-over is the event that leads to
genetic recombination between linked genes.
• Morgan’s conclusion was that during meiosis, alleles of
some genes assort together because they lie near each
other on the same chromosome.
• In many cases, two alleles inherited from one parent
show a strong tendency to stay together as do those
from the other parent. This phenomenon is called
linkage.
• The closer two genes are on the chromosome, the more
likely they are to remain together during meiosis.
 Construction of genetic maps
• Genetic mapping is the process that uses genetic
experiment to determine the relative position of genes
on chromosomes in eukaryotic organisms.
• A genetic map gives the order in which genes are
arranged along a chromosome and the spacing between
them on the same chromosome.
• The map distance between two genes is based on the
frequency of recombination between the two genes.
The recombination frequency is an approximation of
the frequency of crossovers between the two genes.
• The frequency of crossing-over, and hence
recombinants, for linked genes is characteristic of the
gene pairs involved e.g. for the white eye (w) and
miniature wing (m) which are X-linked the frequency
of crossing-over is 36.9%
• The frequency of recombinants for two linked
genes is the same regardless of how alleles of
the two genes involved are arranged on
homologous chromosomes.
• In an individual doubly heterozygous for the w
and m alleles, the alleles can be arranged in two
ways; an arrangement called coupling (or the cis
configuration) w+m+/wm in which the two wild
type alleles are on one homolog or an
arrangement called repulsion (or the trans
configuration) w+m/wm+ in which each
homolog carries the wild-type allele.
Genetic mapping using two-point testcrosses

• By carrying out two-point testcrosses we can determine

the relative numbers of parental and recombinant
classes.
• For autosomal recessive genes, a double homozygous
wild individual is crossed with a doubly homozygous
recessive mutant strain i.e. a+b+/a+b+ x ab/ab to give
a+b+/ab F1. The F1 a+b+/ab is testcrossed with ab/ab.
Four phenotypic classes are found in the F2, two have
the parental phenotypes a+b+ and ab and the other two
have recombinant phenotypes a+b and a b+.
• The parental classes result from diploids in which no
crossover occurred and they occur in approximately
equal numbers.
• The recombinant classes derive from diploids in which
a single crossover event has occurred between
homologous chromosomes. They also occur in
approximately equal numbers.
• Because a single crossover event occurs more rarely
than no crossing-over, an excess of parental phenotypes
over recombinants in the progeny of a testcross
indicates linkage between the genes involved.
• For X-linked recessive genes, a double heterozygous
female is testcrossed with a hemizygous male carrying
the recessive allele (i.e. a+b+/ab x ab/Y).
• For any testcross, the percentage of recombinants in the
progeny cannot exceed 50%. If the genes are assorting
independently, an equal number of recombinants and
parentals are expected, hence the frequency of
recombinants is 50% and the genes involved are
unlinked.
• Genes may be unlinked in two ways: the genes may be
on different chromosomes or the genes may be on the
same chromosome but lie far apart that at least one
crossover is certain to occur between them.
• The characteristic crossover frequencies for linked
genes can be related to the physical distances
separating the genes on the chromosome.
• The percentage of recombinant is used as a quantitative
measure of the genetic distance between two gene pairs
on a genetic map.
• The distance between two genes is measured in map
units. The map unit (mu) is also called a centimorgan
(cM).
• A crossover frequency of one percent between two
genes is defined as one map unit. That is, one map unit
is the distance between gene pairs for which one
product out of 100 (1%) is recombinant.
• Before beginning any experiment to construct a genetic
map, geneticists must show that the genes under
consideration are linked.
• A way to test for linkage is to analyze the results of
crosses to see whether the number of offspring in
different phenotypic classes deviate significantly from
the ratios expected from independent assortment of
genes.
• The best cross to use is the testcross, a cross of one
individual with unknown genotype with another
individual homozygous recessive for all genes
involved.
• A chi-square (χ2) test can be used to determine if the
observed numbers significantly deviate from the
expectation.
• In Drosophila, b is a recessive autosomal mutation
which, when homozygous, results in black body
colour, and vg is a recessive autosomal mutation
which, when homozygous, results in flies with
vestigial (short, crumpled) wings. Wild-type flies
have grey bodies and normal (long, uncrumpled)
wings. True-breeding black, normal wing (b/b
vg+/vg+) flies were crossed with true-breeding
grey, vestigial wing (b+/b+ vg/vg) flies. F1 grey,
normal (b+/b vg+/vg) female flies were testcrossed
to black, vestigial (b/b vg/vg) male flies. The
testcross progeny were:
- 283 grey-normal, 1,294 grey-vestigial, 1,418
black-normal and 241black-vestigial.
- Use the χ2 test to test the hypothesis that the two
genes are unlinked.
• If the two genes are unlinked, then a testcross
should result in a 1:1:1:1 ratio of the four
phenotypic classes.
• A significant deviation from this ratio in the
direction of too many parental types and too few
recombinant types would suggest that the two
genes are linked.
• The chi-square test can be used to make a decision
on the significance of the deviation. The
calculation of chi-square is shown in the Table
below. Given the total number of progeny (3,236)
and the hypothesis under evaluation (1:1:1:1
ratio), the expected number of progeny for each
phenotypic class is 809. The chi-square value is
χ2 = ΣD2/E; where D is the deviation(O-E) and E
is the expected value.
 Calculation of chi-square

Phenotypes Observed Expected Deviation (D) D2 D2/E

number (O) number (E)
Grey-normal 283 809 -526 276,676 342.00

Grey-vestigial 1,294 809 485 235,225 290.76

Back-normal 1,418 809 609 370,881 458.44

Black-vestigial 241 809 -568 322,624 398.79

Total 3,236 3,236 χ2 = 1,489.99

• The χ2 value and the degree of freedom (df) (n-1,
n being the number of phenotypic classes) are
used with a table of χ2 probabilities to determine
the probability (P) that the deviation of the
observed values from the expected values is due to
chance. If P is greater than 5% (P > 0.05), the
deviation is statistically not significant. If P is less
or equal to 5% (P ≤ 0.05), the deviation is
statistically significant, not due to chance alone.
• For our example, χ2 = 1,490, with 3 df, the P value
is much lower than 0.001. Thus, we reject the
hypothesis of independent assortment, and think
of an alternative hypothesis that the genes are
linked.
• Since the genes are linked, we can calculate the
genetic distance between the two genes and
construct a genetic map.
• The map distance between two genes is indirectly
indicated by the frequency of recombination
between the two genes, which is an approximation
of the frequency of crossovers between the two
genes.
• To get the recombination frequency, we calculate
the percentage of recombinant types;
• Percent recombinant =
(number of recombinants /total number of progeny) x 100.
• The value for the percentage of recombinants is
usually converted directly into map units (mu).
• For our example, the recombinants are the grey-
normal (283) and black-vestigial (241) progeny.
The parentals are the grey-vestigial (1,294) and
back-normal (1,418).
• Hence, the number of recombinants is 524 and the
total number of progeny is 3,236.
• Percent recombinants = (524/3,236) x 100 =
16.19%.
• Thus, the two genes are 16.2 mu or 16.2 cM apart.
 Double crossovers
• For low recombination fraction (less than 10%), 1
map unit (1 cM) equals 1% recombination
frequency. For distance greater than this, there is
an appreciable chance that more than one
crossover will occur between loci.
• If two crossovers occur the event is called a
double crossover.
• A single crossover between linked genes generates
half parental and half recombinant gametes
whereas a double crossover gives only parental
types.
Effects of double crossovers in genetic mapping

• Double crossovers does not result in

recombination of the allelic pairs, so only parental
progeny are produced. The same progeny would
result if there was no crossing over between the
two chromatids.
• The double crossover event does not generate
recombinants, thus the result of the two crossovers
are uncounted.
• Since genetic map distance is derived from the
average frequency of crossing over occurring
between linked genes, the estimate of map
distance between two genes will be low.
• If no multiple crossovers occurs between linked
genes, there will be a direct linear relationship
between genetic map distance and recombination
frequency.
• When genes are far apart, the incidence of
multiple crossovers causes the recombination
frequency to be an underestimate of the genetic
map distance.
• Double crossovers occur rarely within distances of
10 mu or less, and they occur more frequently in
greater physical distances for certain chromosome
regions.
• One way to get accurate map distance is to study
closely linked genes.
• Another efficient way is to use a three point
testcross involving three genes within a relatively
short section of a chromosome.
Mapping genes using three-point test cross

• A three-point testcross is a cross of a triple

heterozygote with a triply homozygous recessive.
• A double crossover between linked genes gives
only parental gametes, thus inaccurate map
distances between genes result, since not all
crossovers can be counted.
• The potential advantage of the three-point
testcross is that the presence of a third allelic pair
between the two genes enables the detection of the
double crossover event. In a double crossover, the
middle gene will change positions relative to the
outside genes (see figure below)
• In the figure above, a two-strand double
crossover event between genes ‘a’ and ‘c’ is
detected by the recombination of the B/b
allelic pair in relation to the other two
allelic pairs.
• If the mutant genes in the cross are all
recessive, a typical three-point testcross will
be: a+b+c+/abc x abc/abc.
• In a testcross involving sex-linked genes,
the female is the heterozygous strain
(assuming that the female is the
homogametic sex) and the male is
hemizygous for the recessive alleles.
 Example
• In Drosophila the non-wild type alleles for eye
colour, cross-wing vein and wing edge are v
(vermilion eyes), cv (cross veinless wings) and ct
(cut wing edges), respectively. A cross was made
between the homozygous doubly recessive flies of
genotype v+ cv ct/v+ cv ct and homozygous singly
recessive flies of genotype v cv+ ct+/v cv+ ct+.
From this cross triply heterozygous progeny of
genotype v cv ct/v+ cv+ ct+ were obtained and the
females of this genotype were testcrossed to triple
recessive males of genotype v cv ct/v cv ct. The
progeny types and their numbers are shown below.
From these data calculate the map distance
between genes.
Class Genotype Number observed
1 v cv+ ct+ 580
2 v+ cv ct 592
3 v cv ct+ 45
4 v+cv+ ct 40
5 v cv ct 89
6 v+ cv+ ct+ 94
7 v cv+ ct 3
8 v+ cv ct+ 5
• Step 1: Determine the parental genotypes,
recombinant genotypes from single crossover
and double crossover
- The most frequently observed classes (i.e class 1
and 2) are the parental types. Parental
genotypes are v cv+ ct+ and v+ cv ct.
- The frequency of a double crossover in a region
is expected to be lower than the frequency of a
single crossover, hence, double-crossover
genotypes are the least frequent class found.
Class 7 and 8 are recombinants from double
crossover gametes.
- Class 3 to 6 are recombinants from single
crossover gametes.
• Step 2: Determine the gene order
- To determine the gene order we must first
determine which gene is in the centre.
- Recall that a double crossover event involving the
same two chromatids changes the orientation of
the gene in the centre of the sequence with respect
to the two flanking allelic pairs.
- By comparing the parental genotypes (v cv+ ct+
and v+ cv ct) and the genotypes of double
crossover gametes (v cv+ ct and v+ cv ct+), we
see that the gene that changed its orientation is ct
and this gene must be in the centre.
- Therefore, the gene order is v ct cv. The gene
order for all classes can now be rewritten as
follows:
Class Genotypes Number Type
observed
1 v ct+ cv+ 580 Parentals
2 v+ ct cv 592 Parentals
3 v ct+ cv 45 Recombinants (single crossovers in
region ct – cv)
4 v+ ct cv+ 40 Recombinants (single crossovers in
region ct – cv)
5 v ct cv 89 Recombinants (single crossovers in
region v – ct)
6 v+ ct+ cv+ 94 Recombinants (single crossovers in
region v – ct)
7 v ct cv+ 3 Recombinants (double crossovers)
8 v+ ct+ cv 5 Recombinants (double crossovers)
• Map distances can now be calculated. The frequency
of crossover is computed between two genes at a
time.
• Recombination frequency between gene v and ct =
(single crossovers in region v-ct + double crossovers/ total number of progeny) x 100
• = ((89 + 94 + 3+5)/1448)x 100 = 13.2%
• The map distance between genes v and ct is 13.2 mu
• Recombination frequency between gene ct and cv =
(single crossovers in region ct-cv + double crossovers/ total number of progeny) x 100
• = ((45 + 40 + 3+5)/1448)x 100 = 6.4%
• The map distance between genes ct and cv is 6.4 mu
• To compute the distance between v and cv we add the two
map distances (v–ct + ct-cv), which is 13.2 + 6.4 = 19.6 mu
• We can now construct the genetic linkage map for the three
genes
Genetic linkage map of the v ct cv region of the chromosome
 Interference and Coincidence
• The map distances can tell us about the
chromosomal recombination. The map distance of
13.2 mu between genes v and ct means that 13.2%
of the gametes should result from crossing-over
between the two gene loci. Similarly, the map
distance of 6.4 mu between genes ct and cv means
that 6.4% of the gametes should result from
crossing-over between the two gene loci.
• However, double crossover progeny typically do
not appear as often as the map distances between
genes lead us to expect.
• Once a crossing-over event has occurred in part of
the meiotic tetrad, the probability of another
crossing-over event occurring nearby is reduced.
• This is probably by physical interference caused
by the breaking and rejoining of the chromatids.
• This phenomenon is called chiasma interference or
chromosomal interference.
• The interference is expressed as a coefficient of
coincidence.
• Coefficient of coincidence = observed double
crossover frequency/expected double crossover
frequency.
• Interference = 1 - Coefficient of coincidence.
• To compute the expected double crossover
frequency we use the product rule: the probability
of two independent events occurring
simultaneously is equal to the product of the
individual probabilities of two single events.
• Thus, the probability of two crossovers (double
crossover) occurring between the genes is equal to
the product of the probabilities of the two events
occurring separately.
• Expected double crossover frequency = (map
distance for region I/100) x (map distance for
region II/100).
• In our example, expected double crossover
frequency = (13.2/100) x (6.4/100) = 0.0084.
• Observed double crossover frequency =
(3+5)/1448 = 0.0055.
• The coefficient of coincidence is 0.0055/0.0084 =
0.655.
• The interference is 1 – 0.655 = 0.345.
• A coincidence of one means that in a given
region all double crossovers occurred that
were expected on the basis of two
independent events, there is no interference,
so the interference value is zero.
• If the coefficient of coincidence is zero,
none of the expected double crossovers
occurred, there is total interference, so the
interference value is one.
• In the example above, the interference value
is 0.345 (i.e. 34.5%), meaning that only
65.5% of the expected crossovers occurred
in the cross.
 Positive or Negative
Interference
• Interference reduces the expected number
of multiple crossovers when a crossover
event in one region of the chromosome
inhibits a second event nearby.
• Interference is positive if fewer double-
crossover events than expected occur and
negative if more double-crossover events
than expected occur.