Linkage maps based on data from Drosophila crosses

Theory:
• In 1903, Walter Sutton and Boveri independently proposed a biological basis for
Mendel’s principles, called the chromosome theory of heredity. The Mendelian “unit
factors” are genes located on chromosomes and hence it is the chromosome, and not
the gene, which is the unit of transmission during meiosis. Most chromosomes contain
a very large number of genes. The genes that are part of the same chromosome
are said to be linked and demonstrate linkage in genetic crosses.

• Because the chromosome, not the gene, is the unit of transmission during meiosis,
linked genes are not free to undergo independent assortment. This tendency of linked
genes to get transmitted together during meiosis is called linkage.

• Genes occasionally switch from one homologous chromosome to the other through
the process of crossing over during the first meiotic prophase, when homologs are
paired, or synapsed, a reciprocal exchange of chromosome segments may take place.

©Richa Misra
 Bateson and Punnett’s experiment with sweet
peas. The genetic cross results in the F2 indicate that
the genes for flower color and pollen length do not
assort independently as excess of parental
phenotypes were observed.

William Bateson and Reginald C. Punnett

©Richa Misra
• Crossing over results in the reshuffling, or recombination, of the alleles between
homologs and always occurs during the tetrad stage. Linkage between genes is
broken down by this recombination (crossing over), and the amount of
recombination between genes is directly related to the distance between them.

• The frequency of crossing over between any two loci on a single chromosome is
proportional to the distance between them, known as the interlocus distance. Thus,
depending on which loci are being considered, the percentage of recombinant
gametes varies. This correlation allows us to construct chromosome maps, which
indicate the relative locations of genes on the chromosomes.
©Richa Misra
A testcross reveals the effects of linkage.

• A testcross for two independently assorting genes is expected to produce a 1:1:1:1

phenotypic ratio in the progeny, with half recombinant progeny and half non-
recombinant progeny.
• With complete linkage, only non-recombinant (parental) progeny are produced.
• Genes that exhibit crossing over are incompletely linked. When linked genes undergo
some crossing over, the result is mostly nonrecombinant progeny and fewer
recombinant progeny.

Linkage Groups
All the genes located on a particular chromosome, form a linkage group. Since the genes
present on a particular chromosome have their alleles located on its homologous
chromosome, the number of linkage groups corresponds to the number of haploid
chromosomes found in a species.

For example, in human being the number of chromosomes are 22 autosomes + 2 sex
chromosomes so the number of linkage groups in males= 22+ 2(one X and one Y) and in
females it is 22 +1 (one X).

©Richa Misra
In 1911, Thomas Hunt Morgan and his student Alfred H. Sturtevant demonstrated in
Drosophila that genes can be mapped by determining the rates of recombination between
the genes. Morgan’s work showed that genes for body color and wing size in Drosophila are
linked and thus don’t assort independently. He proposed that the strength of linkage
between two genes depends upon the distance between the genes on the chromosome.

The procedure for mapping chromosomes was invented by Alfred Sturtevant. He realized
Morgan’s idea and thought if the frequency of crossing over was related to distance, one
could use this information to map out the genes on a chromosome. He derived partial
map of a Drosophila chromosome using Morgan’s data on recombinant frequencies of 6
recessive traits. He used an arbitrary unit of distance-the map unit, or centimorgan (cM)-
equivalent to recombinant frequency of 1%. This methodology laid the foundation for all
subsequent efforts to study the organization of genes in chromosomes.

©Richa Misra
Recombination frequencies can be used to create genetic maps.

Calculating Recombination Frequency

The percentage of recombinant progeny produced in a cross is called the

recombination frequency , which is calculated as follows:

Or R.F. = Single Cross Overs + Double Cross Overs x 100%

Total number of progeny

Since genes are arranged linearly on chromosomes, the probability of a crossover

occurring between 2 genes increases with the distance separating them.

• Distances on genetic maps are measured in map units (abbreviated m.u.);

• one map unit equals 1% recombination.
• Genetic distances measured with recombination rates are approximately additive.

©Richa Misra
Types of Crossing Over
Depending on the number of chiasma, crossing over is of the following types:

Single crossover (SCO)- When chiasma formation takes place at a single point of a
chromosome pair, it is known as single crossing over. In this type two crossed over
chromatids and two non-crossed over chromatids are formed. During meiosis, a limited
number of gene crossover events occur in each tetrad. The frequency of recombinant
gametes is half the frequency of crossing over, and the maximum frequency of
recombinant gametes is 50%.

Double crossover (DCO)- When chiasmata (plural of chiasma) are formed at two
places in the same chromosomes then it is known as double crossing over. In the double
crossing over, formation of each chiasma is independent of the other with the possibility
of upto four types of recombinants. To study a double exchange, 3 gene pairs must be
investigated each heterozygous for 2 alleles. In case of a DCO, 2 separate and
independent events of exchanges must occur simultaneously.
The mathematic probability of 2 independent events occurring simultaneously is equal to
the product of individual probabilities product alone.

So, we can calculate expected frequency of DCO as follows:

Expected DCO= P (crossover at 1 pt.) X P (crossover at 2 pt.) X Total no. of progeny
©Richa Misra
©Richa Misra
Interference & Coefficient of Coincidence
Crossovers are frequently not independent events: the occurrence of one crossover tends
to frequently inhibit additional crossovers in the same region of the chromosome.

The degree to which one crossover interferes with additional crossovers in the same region
is termed the interference (I).

To calculate the interference, we first determine the coefficient of coincidence, which is

the ratio of observed double crossovers to expected double crossovers:

Interference= 1-C.O.C

i. Positive Interference: The occurrence of one tends to inhibit additional crossovers

in the same region of the chromosome and so double crossovers are less frequent
than expected.
ii. Negative Interference: Sometimes more double crossover progeny appear than
expected, which happens when a crossover increases probability of another crossover
nearby.
iii. When interference is complete and no double crossover progeny are observed,
COC is 0 and Interference is 1. ©Richa Misra
Significance of Linkage:
(i) Linkage plays an important role in determining the nature of scope of
hybridization and selection programmes.
(ii) Linkage reduces the chance of recombination of genes and thus helps to hold
parental characteristics together. It thus helps organism to maintain its parental, racial
and other characters. For this reason plant and animal breeders find it difficult to
combine various characters.

Gene Mapping: the determination of the sequence of genes and their relative distances
from one another on a specific chromosome. Gene mapping can be done using genetic
approach (linkage/ recombination data) or by physical methods (restriction mapping).

Genetic maps are useful in many ways:

i) They allow to understand the overall complexity and genetic organization of a
particular species.
ii) Improve our understanding of the evolutionary relationships among different
species.
iii) Provide helpful information for improving agricultural traits.
iv) Can offer firm evidence that a disease transmitted from parent to child is linked to
one or more genes.

©Richa Misra
Constructing a Genetic Map with the Use of Two-Point Testcrosses

A testcross between two genes is called a two-point testcross or a two-point cross for
short.
Genetic maps can be constructed from a series of testcrosses for pairs of genes, but
this approach is not particularly efficient, because numerous two-point crosses must be
carried out to establish the order of the genes and because double crossovers are
missed.

A more efficient mapping technique is a testcross for three genes—a three-point

testcross , or three-point cross. With a three-point cross, the order of the three genes
can be established in a single set of progeny and some double crossovers can usually be
detected, providing more-accurate map distances.

©Richa Misra
Steps required to map genes from a three-point cross:

1. Write out the phenotypes and numbers of progeny produced in the three-point
cross.

2. Write out the genotypes of the original parents used to produce the triply
heterozygous individual in the testcross.

3. Determine which phenotypic classes among the progeny are the non-recombinants
and which are the double crossovers. The non-recombinants will be the two most-
common phenotypes; double crossovers will be the two least-common phenotypes.

4. Determine which locus lies in the middle. Compare the alleles present in the double
crossovers with those present in the non-recombinants; each class of double
crossovers should be like one of the non-recombinants for two loci and should
differ for one locus. The locus that differs is the middle one.

5. Rewrite the phenotypes with the genes in correct order.

6. Determine where crossovers must have taken place to give rise to the progeny
phenotypes. To do so, compare each phenotype with the phenotype of the non-
recombinant progeny. ©Richa Misra
7. Determine the recombination frequencies. Add the numbers of the progeny that
possess a chromosome with a crossover between a pair of loci. Add the double
crossovers to this number. Divide this sum by the total number of progeny from the
cross, and multiply by 100%; the result is the recombination frequency between the
loci, which is the same as the map distance.

8. Draw a map of the three loci. Indicate which locus lies in the middle, and indicate the
distances between them.

9. Determine the coefficient of coincidence and the interference. The coefficient of

coincidence is the number of observed double-crossover progeny divided by the
number of expected double-crossover progeny. The expected number can be
obtained by multiplying the product of the two single recombination probabilities by
the total number of progeny in the cross.

©Richa Misra
Question 1: In Drosophila melanogaster, scarlet eyes (st)
are recessive to wild-type red eyes (st+), ebony body
color (e) is recessive to wild-type gray body color
(e+), and spineless (ss)—that is, the presence of small
bristles—is recessive to wild-type normal bristles
(ss+). The loci encoding these three characteristics are
linked and located on chromosome 3. A map is
obtained by crossing a female who is heterozygous
for all 3 mutations to a male homozygous for all 3
mutations. The data is as follows:

a. Determine the linear order of genes on the

chromosome.
b. Calculate the linkage map.
c. Determine the coefficient of coincidence and
interference.

You can write the data simply instead of drawing

the details like in next questions:
©Richa Misra
Step 1: identifying the parental class (highest frequency) and double crossover progeny class
(lowest frequency)

Parental class: st+ e+ ss+ 283 D.C.O: st+ e+ ss 5

st e ss 278 st e ss+ 3

Step 2: Comparison of parental and DCO class to determine the position of middle gene

Method 1: As ss is the gene that is swapping in position, hence it is the middle gene.
Method 2: Only write once in
Method 2: this question for practice

Three orders of genes on the

chromosome are possible: the eye-color
locus could be in the middle (e st ss ), the
body-color locus could be in the middle (st
e ss), or the bristle locus could be in the
middle (st ss e).

The only gene order that produces chromosomes with the set of alleles observed in the least-
numerous progeny or double crossovers ( st+ e+ ss and st e ss+ in Figure) is the one in which the ss
locus for bristles lies in the middle (gene-order 3). Therefore, this order (st ss e ) must be the correct
sequence of genes on the chromosome. ©Richa Misra
Step 3: Writing given data in order:

st+ ss+ e+ 283

st ss e 278
st+ ss e 50
st ss+ e+ 52
st+ ss+ e 43
st ss e+ 41
st+ ss e+ 5
st ss+ e 3

Step 4: Determine the single crossover between st and ss and their recombinant frequency:

Recombinant progeny that possess a chromosome that underwent crossing over between
the st and ss include the single crossovers (st+/ss e and st /ss+ e+) and the two double
crossovers (st+/ss/e+ and st/ss+/e ).
©Richa Misra
So, Recombination frequency between st and ss is:

Step 5: The map distance between the ss and e is determined in the same manner.

Step 6: Making a map.

Map distance can be obtained by summing the map distances between st and ss and between ss
and e (14.6 m.u. + 12.2 m.u.= 26.8 m.u.). We can now use the map distances to draw a map of the
three genes on the chromosome:

©Richa Misra
Step 7: Expected DCO is calculated

Double crossovers between st and e is equal to the probability of recombination

between st and ss multiplied by the probability of recombination between ss and e , or
0.146 x 0.122 = 0.0178.
Multiplying this probability by the total number of progeny gives us the expected
number of double-crossover progeny from the cross: 0.0178 x 755 = 13.4.

So the interference for our three-point cross is:

interference = 1 − 0.6 = 0.4. This value of interference tells us that 40% of the double-
crossover progeny expected will not be observed, because of interference. So Positive
interference.

©Richa Misra
Question 2

©Richa Misra
Answer:

Don’t skip steps

Do in sequence to
avoid any error

©Richa Misra
Question 3: In Drosophila, a heterozygous female for the X-linked recessive traits
a, b, and c was crossed to a male that phenotypically expressed a, b, and c. The
offspring occurred in the following phenotypic ratios:

No other phenotypes were observed.

(a) What is the genotypic arrangement of the alleles of these genes on the X
chromosome of the female?
(b) Determine the correct sequence and construct a map of these genes on the X
chromosome.
(c) What progeny phenotypes are missing? Why?

©Richa Misra
Answer:

Write with Steps, not shortcut like here

©Richa Misra
Question 4: In Drosophila, a cross was made between females—all expressing the three
X-linked recessive traits scute bristles (sc), sable body (s), and vermilion eyes (v)—and
wild-type males. In the F1, all females were wild type, while all males expressed all three
mutant traits. The cross was carried to the F2 generation, and 1000 offspring were
counted, with the results shown in the following table.

No determination of sex was made in the data.

(a) Using proper nomenclature, determine the genotypes of the P1 and F1 parents.
(b) Determine the sequence of the three genes and the map distances between them.
(c) Are there more or fewer double crossovers than expected?
(d) Calculate the coefficient of coincidence. Does it represent positive or negative
interference?
©Richa Misra
©Richa Misra