Multigenic Traits

The genes of an individual do not operate isolated from one another, but obviously are
functioning in a common cellular environment. Thus, it is expected interactions between
genes would occur. Bateson and Punnett performed a classical experiment that
demonstrated genetic interactions. They analyzed the three comb types of chicken
known to exist at that time:

Chicken Varieties

Phenotype

Wyandotte

Rose Comb

Brahmas

Pea Comb

Leghorns

Single Comb

Rose Pea
Single Walnut
Result: The F1 differed from both parents and two new phenotypes not seen in the
parents appeared in the F2. How can this result be explained? The first clue is the F 2
ratio. We have seen this ratio before when the F1 from a dihybrid cross is selfed (or
intermated). This observation suggests that two genes may control the phenotype of the
comb. The gene interactions and genotypes were determined by performing the
appropriate testcrosses.

A series of experiments demonstrated that the genotypes controlling the various comb
phenotypes are as follows.

Phenotypes

Genotypes

Frequency

Walnut

R_P_

9/16

Rose

R_pp

3/16

Pea

rrP_

3/16

Single

rrpp

1/16

It was later shown that the genotypes of the initial parents were:

Rose = RRpp

and

Pea = rrPP

Therefore, genotypically the cross was:

The development of any individual is obviously the expression of all the genes that are a
part of its genetic makeup. Therefore, it is not an unexpected conclusion that more than
one gene could be responsible for the expression of a single phenotype. We will now
discuss this situation. First let's give a definition.
Epistasis - the interaction between two or more genes to control a single phenotype
The interactions of the two genes which control comb type was revealed because we
could identify and recognize the 9:3:3:1. Other genetic interactions were identified
because the results of crossing two dihybrids produced a modified Mendelian ratio. All
of the results are modifications of the 9:3:3:1 ratio. These ratios also indicate that these
genes reside on different chromosomes (and therefore assort independently).

Duplicate gene interaction (F2 expected ratio, 15:1 Ratio)

In this type of interaction, two different genes code each for an enzyme that catalyzes
the same reaction. Therefore, regardless if one of them is disfunctional, the product of
the other will still catalyze the reaction and provide the wild-type phenotype.

Example: Kernel Color in Wheat

For this type of pathway a functional enzyme A or B can produce a product from a
common precursor. The product gives color to the wheat kernel. Therefore, only one
dominant allele at either of the two loci is required to generate the product.
Thus, if a pure line wheat plant with a colored kernel (genotype = AABB) is crossed to
plant with white kernels (genotype = aabb) and the resulting F1 plants are selfed, a
modification of the dihybrid 9:3:3:1 ratio will be produced. The following table provides a
biochemical explanation for the 15:1 ratio.
[Notice that the "biology" behind the kernel colouring is what impacts the phenotypic
ratio in the F2 following a Mendelian cross]
Genotyp
e

Kernel
Phenotype

Enzymatic
Activities

9 A_B_

colored
kernels

functional enzymes
from both genes

3 A_bb

colored
kernels

functional enzyme
from the A gene
pair

3 aaB_

colored
kernels

functional enzyme
from the B gene
pair

1 aabb

colorless
kernels

non-functional
enzymes produced
at both genes

If we sum the three different genotypes that will produce a colored kernel we can see
that we can achieve a 15:1 ratio. Because either of the genes can provide the wild type
phenotype, this interaction is called duplicate gene action.

Complementary gene interaction (F2 expected ratio, 9:7 Ratio)

Example: Flower color in sweet pea
If two genes are involved in a specific pathway and functional products from both are
required for expression, then one recessive allelic pair at either allelic pair would result
in the mutant phenotype. This is graphically shown in the following diagram.

Notice that both precursors in this pathway have the same

phenotype. Therefore, a dominant allele at locus is
necessary to have the other phenotype.How does that
influence Mendelian ratios associated with a dihybrid cross.?

If a pure line pea plant with colored flowers (genotype = CCPP) is crossed to pure line,
homozygous recessive plant with white flowers, the F 1 plant will have colored flowers
and a CcPp genotype. The normal ratio from selfing dihybrid is 9:3:3:1, but epistatic
interactions of the C and P genes will give a modified 9:7 ratio. The following table
describes the interactions for each genotype and how the ratio occurs.

Genotype

Flower Color

Enzyme Activities

9 C_P_

Flowers colored;
anthocyanin produced

Functional enzymes from both genes

3 C_pp

Flowers white;
no anthocyanin produced

p enzyme non-functional

3 ccP_

Flowers white;
no anthocyanin produced

c enzyme non-functional

1 ccpp

Flowers white;
no anthocyanin produced

c and p enzymes non-functional

Message: Because both genes are required for the correct phenotype,
this epistatic interaction is called complementary gene action.

Dominant Epistasis
Dominant Epistasis (F2 expected ratio, 12:3:1 Ratio)
Example: Fruit Color in Squash

With this interaction, color is recessive to no color at one allelic pair. This recessive
allele must be expressed before the specific color allele at a second locus is expressed.
At the first gene white colored squash is dominant to colored squash, and the gene
symbols are W=white and w=colored. At the second gene yellow is dominant to green,
and the symbols used are G=yellow, g=green. If the dihybrid is selfed, three
phenotypes are produced in a 12:3:1 ratio. The following table explains how this ratio is
obtained.

Shapes of Squash Fruit

Genotype Fruit Color

Gene Actions

9 W_G_

White

Dominant white allele negates effect of G allele

3 W_gg

White

Dominant white allele negates effect of G allele

3 wwG_

Yellow

Recessive color allele allows yellow allele expression

1 wwgg

Green

Recessive color allele allows green allele expression

Message: Because the presence of the dominant W allele masks the effects of either
the G or g allele, this type of interaction is called dominant epistasis.

Recessive Epistasis
Recessive Epistasis (F2 expected ratio, 9:3:4 Ratio)
Example: Labrador coat colour
Another case of recessive epistasis well known to most people is the yellow coat color
of Labrador retriever dogs. Two alleles, B and b, stand for black and brown coats,
respectively, but the allele e of another gene is epistatic on these alleles, giving a yellow
coat. Therefore the genotypes B/;e/e and b/b;e/e are both of yellow phenotype,
whereas B/;E/ and b/b;E/ are black and brown, respectively. This case of epistasis
is not caused by an upstream block in a pathway leading to dark pigment. Yellow dogs
can make black or brown pigment, as can be seen in their noses and lips. The action of
the allele e is to prevent deposition of the pigment in hairs. In this case, the epistatic

gene is developmentally downstream; it represents a kind of developmental target that

has to be of E genotype before pigment can be deposited.
F2 ratio from a dihybrid cross (EeBb X EeBb - both black labs) --> 9 E-B- (Black):
3 Ee-bb (chocolate): 4 [3 eeB- + 1 eebb (yellow)]
Gene suppression (F2 expected ratio, 13:3 ratio)
Example: Malvidin production in Primula
Certain genes have the ability to suppress the expression of a gene at a second locus.
The production of the chemical malvidin in the plant Primula is an example. Both the
synthesis of the chemical (controlled by the K gene) and the suppression of synthesis at
the K gene (controlled by the D gene) are dominant traits. The F1 plant with the
genotype KkDd will not produce malvidin because of the presence of the dominant D
allele. What will be the distribution of the F2 phenotypes after the F1 was crossed?

Genotype

Phenotype and genetic explanation

9 K_D_

no malvidin because dominant D allele is present

3 K_dd

malvidin productions because dominant K allele present

3 kkD_

no malvidin because recessive k and dominant D alleles present

1 kkdd

no malvidin because recessive k allele present

The ratio from the above table is 13 no malvidin production to 3 malvidin production.
Because the action of the dominant D allele masks the genes at the K locus, this
interaction is termed dominant suppression epistasis.
Suppressor - a genetic factor that prevents the expression of alleles at a second locus;
this is an example of epistatic interaction.

- Impact of Gene Interactions on Mendelian ratios

Other Types of Epistatic Interactions

Today, scientists know that Mendel's predictions about inheritance depended on the genes he
chose to study. Specifically, Mendel carefully selected seven unlinked genes that affected
seven different traits. However, unlike the phenotypes that Mendel considered, the majority of
phenotypes are affected by more than one gene. Indeed, most of the characteristics of
organisms are much more complex than the characteristics that Mendel studied, and epistasis
is one source of this complexity. Epistasis can occur in a variety of different ways and result in
a variety of different phenotypic ratios, as illustrated in the Table. Beyond epistasis, geneenvironment interactions further increase the variety of phenotypes we see around us each
day.
Table: Examples of Digenic Epistatic Ratios

Ratio

9:3:3:1

9:4:3

9:7

12:3:1

15:1

13:3

9:6:1

Description

Complete dominance at both gene pairs; new

phenotypes result from interaction between
dominant alleles, as well as from interaction
between both homozygous recessives

Name(s) of Relationship
(Used by Some Authors)

Multigenic Traits

Recessive epistasis
Complete dominance at both gene pairs; however,
when one gene is homozygous recessive, it hides
the phenotype of the other gene
Complete dominance at both gene pairs; however,
when either gene is homozygous recessive, it
hides the effect of the other gene

Complete dominance at both gene pairs; however,

when one gene is dominant, it hides the phenotype
of the other gene

Complementary gene
interaction

Dominant epistasis

Duplicate gene interaction

Complete dominance at both gene pairs; however,
when either gene is dominant, it hides the effects
of the other gene
Gene suppression
Complete dominance at both gene pairs; however,
when either gene is dominant, it hides the effects
of the other gene
Duplicate interaction
Complete dominance at both gene pairs; however,
when either gene is dominant, it hides the effects

of the other gene

7:6:3

3:6:3:4

11:5

No name
Complete dominance at one gene pair and partial
dominance at the other; when homozygous
recessive, the first gene is epistatic to the second
gene
No name
Complete dominance at one gene pair and partial
dominance at the other; when homozygous
recessive, either gene hides the effects of the
other gene; when both genes are homozygous
recessive, the second gene hides the effects of the
first
No name
Complete dominance for both gene pairs only if
both kinds of dominant alleles are present;
otherwise, the recessive phenotype appears

Introduction - What is Gene Linkage

Genetic linkage is the tendency of alleles that are located close together on a
chromosome to be inherited together during meiosis. Genes whose loci are nearer to
each other are less likely to be separated onto different chromatids during chromosomal
crossover, and are therefore said to be genetically linked. In other words, the nearer two
genes are on a chromosome, the lower is the chance of a swap occurring between
them, and the more likely they are to be inherited together.
During meiosis, chromosomes assort randomly into gametes, such that the segregation
of alleles of one gene is independent of alleles of another gene. This is stated in
Mendel's Second Law and is known as the law of independent assortment. The law
of independent assortment always holds true for genes that are located on different
chromosomes, but for genes that are on the same chromosome, it does not always hold
true.

As an example of independent assortment, consider the crossing of the pure-bred

homozygote parental strain with genotype AABB with a different pure-bred strain with
genotype aabb. A and a and B and b represent the alleles of genes A and B. Crossing
these homozygous parental strains will result in F1 generation offspring that are double
heterozygotes with genotype AaBb. The F1 offspring AaBb produces gametes that are
ABam6), aB, and ab with equal frequencies (25%) because the alleles of gene A assort
independently of the alleles for gene B during meiosis. Note that 2 of the 4 gametes
(50%)Ab and aBwere not present in the parental generation. These gametes
represent recombinant gametes. Recombinant gametes are those gametes that differ
from both of the haploid gametes that made up the original diploid cell. In this example,
the recombination frequency is 50% since 2 of the 4 gametes were recombinant
gametes (see figure below).
The recombination frequency will be 50% when two genes are located on different
chromosomes or when they are widely separated on the same chromosome. This is a
consequence of independent assortment.
When two genes are close together on the same chromosome, they do not assort
independently and are said to be linked. Whereas genes located on different
chromosomes assort independently and have a recombination frequency of 50%, linked
genes have a recombination frequency that is less than 50% (see figure below). We
know that recombination between two genes on the same chromosome is the result of
homolougous recombination as a result of crossin-overs betweem non-sister chromatids
of homologous chromosomes during meiosis

Type of recombination

Independent Assortment during meiosis (I)

if (P) AABB X aabb
(F1) AaBb
Note: that 2 of the 4 gametes (50%)Ab and aBwere
not present in the parental generation. These gametes
represent recombinant gametes. Recombinant gametes
are those gametes that differ from both of the haploid
gametes that made up the original diploid cell.

Outcome during meiosis

Homologous recombination due to cross-overs

if (P) AB/AB X ab/ab
(F1) AB/ab
Note: When two genes are close together on the same
chromosome, they do not assort independently and are
said to be linked.
Note: In this figure, homologous recombination does
create the same allelic combination as would be observed
during independent assortment. Given that cross-overs
do NOT always occur in the same locations, linked genes
have a recombination frequency that is less than 50%.

Complete Linkage
if (P) AB/AB X ab/ab
(F1) AB/ab
Note: When two genes are so close together on the
same chromosome, homologous recombinations do not
occur between them. In this case, there no
recombinations. Therefore, we woud see 0%
recombinants.

When two genes are located on the same chromosome, the chance of a crossover
producing recombination between the genes is related to the distance between the two
genes. Thus, the use of recombination frequencies has been used to develop linkage
maps or genetic maps.

Dihybrids: Independant assortment vs Linked Genes

Figure of the types of recombination during meiosis and consequences on the

types of gametes that are formed:
For the following table, assume the parental (P) generation cross is between AABB X
aabb individuals.

2 point mapping
Physical crossing over during meiosis I is a normal event. The effect of this event is to
rearrange heterozygous homologous chromsomes into new combinations. The term
used for crossing over is recombination. Recombination can occur between any two
genes on a chromosome, the amount of crossing over is a function of how close the
genes are to each other on the chromosome. If two genes are far apart, for example at
opposite ends of the chromosome, crossover and non-crossover events will occur in
equal frequency. Genes that are closer together undergo fewer crossing over events
and non-crossover gametes will exceed than the number of crossover gametes. The
figure below shows this concept.
So, 2-point mapping is a strategy to 1) determine if two genes are linked, and if so
2) to determine the recombination frequency to gain an estimate of the distance
between the two genes. This is done by crossing a heterozygote with a homozygous
recessive (2 genes) in a testcross.
Purpose of the testcross? Given that the tester always gives the recessive alleles, the
ratio of the phenotypes of the offspring of the cross will reveal the types of gametes
produced by the F1 heterozygote.
The figure below depicts the gamete composition for linked genes from coupling
and repulsion crosses:
If the parental generation can be AB/AB and ab/ab (coupling) or Ab/Ab and
aB/aB (repulsion). The heterozygote shown on the left is a repulsion phase and on the
right a coupling phase.

Finally, for two genes are right next to each other on the chromosome crossing over will
be a very rare event.

Two types of gametes are possible when following genes on the same chromosomes. If
crossing over does not occur, the products are parental gametes. If crossing over
occurs, the products are recombinant gametes. The allelic composition of parental and
recombinant gametes depends upon whether the original cross involved genes in
coupling or repulsion phase.
The figure below depicts the gamete composition for linked genes from coupling
and repulsion crosses:

It is usually a simple matter to determine which of the gametes are recombinant. These
are the gametes that are found in the lowest frequency. This is the direct result of the
reduced recombination that occurs between two genes that are located close to each
other on the same chromosome. Also by looking at the gametes that are most abundant
you will be able to determine if the original cross was a coupling or repulsion phase
cross. For a coupling phase cross, the most prevalent gametes will be those with two
dominant alleles or those with two recessive alleles. For repulsion phase crosses,
gametes containing one dominant and one recessive allele will be most abundant.
Understanding this fact will be important when you actually calculate a linkage distance
estimate from your data.
The important question is how many recombinant chromosomes will be produced. If the
genes are far apart on the chromosome a cross over will occur every time that pairing
occurs and an equal number of parental and recombinant chromosomes will be
produced. Test cross data will then generate a 1:1:1:1 ratio. But as two genes are closer

and closer on the chromosome, fewer cross over events will occur between them and
thus fewer recombinant chromosomes will be derived. We then see a deviation from the
expected 1:1:1:1 ratio.
How can we decide how close two genes are on a chromosome? Because fewer
crossover events are seen between two genes physically close togehter on a
chromosome, the lower the percentage of recombinant phenotypes will be seen in the
testcross data. By definition, one map unit (m.u.) is equal to one percent
recombinant phenotypes. In honor of the work performed by Morgan, one m.u. is
also called one centimorgan (cM).
Now let's determine the linkage distance between the genes pr and vg. We can actually
make two estimates because we have the results from coupling and repulsion phases
crosses. The coupling phase analyzed a total of 2839 gametes, and of these gametes
305 (151 pr+ vg+ 154 pr vg+) gametes were recombinant. To determine the linkage
distance simply divide the number of recombinant gametes into the total gametes
analyzed. So the linkage distance is equal to 10.7 cM [(305/2839)*100)].
We can also perform the same calculations with the results from the repulsion phase
cross. For this experiment, a total of 2335 gametes were analyzed, and 303 (151 pr+ vg+
+ 154 pr vg) of these were the result of recombination. The estimate of the linkage
distance between pr and vgfrom these experiments is 13.0 cM [(303/2335)*100].
Obviously, we can conclude that the two genes are linked on the same chromosome.
But what is the true linkage distance, the 10.7 cM value from the coupling experiment or
the 13.0 value from the repulsion experiment? Actually neither is correct or wrong.
These again are two estimates. Only by repeating this experiments many times using a
number of different independent crosses can we settle on a value.
Once we have settled on a value, these genes can then be graphically displayed. Let's
say that the true distance between the pr and vg genes is 11.8 cM, that is the average
of our two estimates. We can next display them along a chromosome in the manner
shown below. (Note that it is customary to use the allelic designantions of the mutant
phenotype when drawing these maps.)

The final point that we need to make regards the maximum distance that we can
measure. Because of the way in which the calculations are performed, we can never
have more that 50% recombinant gametes. Therefore the maxmimum distance that two
genes can be apart and still measure that distance is just less that 50 cM. If two genes
are greater than 50 cM apart, then we can not determine if they reside on the same
chromosome or are on different chromosomes. In practice though, when experimental
error is considered, as distances approach 50 cM it is difficult to determine if two genes

are linked on the same chromosome. Therefore, other mapping techniques must be
used to determine thelinkage relationship among distantly associated genes. One
method that allows us to deal with distantly related genes and to order genes is the
three-point cross.

3 point mapping
In genetics, a three-point cross is used to determine the loci of three genes in an
organism's genome. This helps to refine, that is provide or resolve gene map more
accurately.
An individual heterozygous for three mutations is crossed with a homozygous recessive
individual, and the phenotypes of the progeny are scored. The two most common
phenotypes that result are the parental gametes; the two least common phenotypes that
result come from a double crossover in gamete formation. By comparing the parental
and double-crossover phenotypes, the geneticist can determine which gene is located
between the others on the chromosome.
The recombinant frequency is the ratio of non-parental phenotypes to total individuals. It
is expressed as a percentage, which is equivalent to the number of map units (or
centiMorgans) between two genes. For example, if 100 out of 1000 individuals display
the phenotype resulting from a crossover between genes a and b, then the
recombination frequency is 10 percent and genes a and b are 10 map-units apart on the
chromosome.
If the recombination frequency is equal to 50 percent, it means that the genes are
unlinked - they are either located on different chromosomes or are sufficiently distant
from each other on the same chromosome. Any recombination frequency greater
than 50 percent is expressed as exactly 50 percent because, being unlinked, they are
equally as likely as not to be separated during gamete formation. To resolve if two
genes are on the same chromosome but very far apart so as to always have a crossingover between then we can add another gene to the analysis in attempt to analyze genes
that are closer to together to pinpoint the map (even for those genes far apart).
By adding a third gene, we now have several different types of crossing over products
that can be obtained. The following figure shows the different recombinant products that
are possible.

Now if we were to perform a testcross with F1, we would expect a 1:1:1:1:1:1:1:1 ratio.
As with the two-point analyzes described above, deviation from this expected ratio
indicates that linkage is occurring. The best way to become familiar with the analysis of
three-point test cross data is to go through an example. We will use the arbitrary
example of genes A, B, and C. We first make a cross between individuals that are
AABBCC and aabbcc. Next the F1 is testcrossed to an individual that is aabbcc. We will
use the following data to determine the gene order and linkage distances. As with the
two-point data, we will consider the F1 gamete composition.
Genotype
ABC

Observed
390

Type of Gamete
Parental

abc

374

Parental

AbC

27

Single-crossover between genes C and B

aBc

30

Single-crossover between genes C and B

ABc

Double-crossover

abC

Double-crossover

Abc

81

Single-crossover between genes A and C

aBC

85

Single-crossover between genes A and C

Total

1000

The best way to solve these problems is to develop a systematic approach. First,
determine which of the the genotypes are the parental gentoypes. The genotypes
found most frequently are the parental genotypes. Why? Because these genes
are linked and are inherited together more often than not. As we started with a
parental line with a given genotype, we can follow this into the F1 and any new
combinations made in the gametes of the F1 would be considered recombinants.

From the table it is clear that the ABC and abc genotypes were the parental genotypes.
Next we need to determine the order of the genes. Once we have determined the
parental genotypes, we use that information along with the information obtained from
the double-crossover. The double-crossover gametes are always in the lowest
frequency. From the table the ABc and abC genotypes are in the lowest frequency. The
next important point is that a double-crossover event moves the middle allele
from one sister chromatid to the other. This effectively places the non-parental allele
of the middle gene onto a chromosome with the parental alleles of the two flanking
genes. We can see from the table that the C gene must be in the middle because the
recessive c allele is now on the same chromosome as the A and B alleles, and the
dominant C allele is on the same chromosome as the recessive a and b alleles.
Now that we know the gene order is ACB, we can go about determining the linkage
distances between A and C, and C and B. The linkage distance is calculated by dividing
the total number of recombinant gametes into the total number of gametes. This is the
same approach we used with the two-point analyses that we performed earlier. What is
different is that we must now also consider the double-crossover events. For these
calculations we include those double-crossovers in the calculations of both interval
distances.
So the distance between genes A and C is 17.9 cM [100*((81+85+5+8)/1000)], and the
distance between C and B is 7.0 cM [100*((27+30+5+8)/1000)].
Now let's try a problem from Drosophila, by applying the principles we used in the above
example. The following table gives the results we will analyze.
Genotype

Observed

Type of Gamete

v cv+ ct+

580

Parental

v+ cv ct

592

Parental

v cv ct+

45

Single-crossover between genes ct and cv

v+ cv+ ct

40

Single-crossover between genes ct and cv

v cv ct

89

Single-crossover between genes v and ct

v+ cv+ ct+

94

Single-crossover between genes v and ct

v cv+ ct

Double-crossover

v+ cv ct+

Double-crossover

Total

1448

Step 1: Determine the parental genotypes.

The most abundant genotypes are the partenal types. These genotypes are v cv+ ct+
and v+ cv ct. What is different from our first three-point cross is that one parent did not
contain all of the dominant alleles and the other all of the recessive alleles.
Step 2: Determine the gene order
To determine the gene order, we need the parental genotypes as well as the double
crossover geneotypes As we mentioned above, the least frequent genotypes are the
double-crossover geneotypes. These geneotypes are v cv+ ct and v+ cv ct+. From this
information we can determine the order by asking the question: In the double-crossover
genotypes, which parental allele is not associated with the two parental alleles it was
associated with in the original parental cross. From the first double crossover, v cv+ ct,
the ct allele is associated with the v and cv+ alleles, two alleles it was not associated
with in the original cross. Therefore, ct is in the middle, and the gene order is v ct cv.
Step 3: Determing the linkage distances.
o v - ct distance caluculation. This distance is derived as follows:
100*((89+94+3+5)/1448) = 13.2 cM
o ct - cv distance calculation. This distance is derived as follows:
100*((45+40+3+5)/1448) =6.4 cM
Step 4. Draw the map.

Three-point crosses also allows one to measure interference (I) among crossover
events within a given region of a chromosome. Specifically, the amount of double
crossover gives an indication if interference occurs. The concept is that given specific
recombination rates in two adjacent chromosomal intervals, the rate of doublecrossovers in this region should be equal to the product of the single crossovers. In the
v ct cv example described above, the recombination frequency was 0.132 between
genes v and ct, and the recombination frequency between ct andcv was 0.064.
Therefore, we would expect 0.84% [100*(0.132 x 0.64)] double recombinants. With a
sample size of 1448, this would amount to 12 double recombinants. We actually only
detected 8.
To measure interference, we first calculate the coefficient of coincidence (c.o.c.)
which is the ratio of observed to expected double recombinants. Interference is then
calculated as 1 - c.o.c. The formula is as follows:

For the v ct cvdata, the interference value is 33% [100*(8/12)].

Most often, interference values fall between 0 and 1. Values less than one indicate that
interference is occurring in this region of the chromosome.