Lab I: Three-Point Mapping in ​Drosophila melanogaster

Makuo Aneke

Partner: Christina Hwang

BIO 365-004: Genetics with Laboratory

TA: Dr. Hongmei Ma

February 18, 2016

 Abstract

The purpose of this experiment was to determine the relationship between three traits

​ found in ​Drosophila melanogaster, also known as fruit flies. The hypothesis for the experiment

​ was​ that the traits studied

​ (wing size (​m), eye color (​w), and bristle type (​f)) were X-linked and

not independently assorted. This was done by breeding the fruit flies to obtain three generations

(P​1​, F​1​, and F​2​) that will aid in determining the mode of inheritance. Results from the experiment

led to the conclusion that the three genes were X-linked. The gene order determined was ​wmf

​ ​ and the map distances were

​ ​
44.6 m.u. (between ​w and ​m), 37.8 m.u. (between ​m and ​f), and

~70.4 m.u. (between ​w and ​f). The linkage map constructed correlated fairly with the

recombination frequencies, however there were discrepancies involving the frequency of single

crossovers.
 Introduction

One of the most important organisms that have been used to study genetics for several

decades is ​Drosophila melanogaster, also called the “fruit fly,” known to spontaneously arise in

the presence of ripened fruit. They are very useful for genetic analysis because they breed easily,

they have short lifespans and reproduction periods, and they’re easy to maintain and control in

regards to food and temperature. Also, fruit flies are small enough to collect large populations

yet large enough to distinguish wild type traits from mutant traits (Klug et. al. 2012).

​ The specific traits studied in this experiment are wing size (​m), eye color (​w), and types

of bristles (​f). Wild type fruit flies have a longer wing size (extending past the abdomen), red eye

color, and long, sleek bristles seen on the posterior abdomen. Mutant fruit flies have short wings

(not extending past the abdomen), white eye color, and forked/bent bristles (often the hardest to

distinguish). These traits are known to be X-linked in ​Drosophila melanogaster rather than

independently assorted. Unlike independently assorted genes, linked genes are located on

homologous chromosomes and their locations on the chromosome influence heredity (Klug et.

al. 2012). X-linked genes are traits that are exclusive to the X sex chromosome and often creates

sex bias in offspring (Klug et. al. 2012). Other characteristics that are associated with linked

genes are three-point crosses, map distances, and gene order. When three loci are involved,

three-point crosses are used to diagram the number of offspring for each phenotype and the

frequency of recombination between the two homologous chromosomes ​(Aggarwal et. al. 2015)​.

The number of offspring are used to determine the order of the genes by calculating the

frequency of parental genotypes (without any crossing over) single crossovers (between any two

neighboring traits) and double crossovers (between two neighboring traits simultaneously).
Normally with linked traits, parental genotypes are most frequent while double crossovers are

least frequent ​(Aggarwal et. al. 2015)​.

The chance of recombination directly correlates with map distance, meaning that traits

that are farther apart are more likely to participate in crossing over ​(Aggarwal et. al. 2015)​. It is

calculated by dividing the recombination events (between two loci) by the total number of

events. Once the chance of recombination is determined, it can automatically be used to

determine distances between the genes (chance of recombination, % = map distance, m.u.).

These values can then be used to construct a gene map to visualize where the genes are located

on the chromosome (Klug et. al. 2012).

The hypothesis for this experiment was that the genes studied in this experiment (​wfm)

are X-linked and not independently assorted. The strategy was to successfully mate these fruit

flies in order to determine linkage of mutant and wild type traits. The goal was to produce

enough offspring in each generation to breed a large enough sample size in order to obtain more

accurate results.
Methods and Materials

The experiment began with the use of two fly stocks: wild type males (+++) and fully

mutant females (​wfm). The ​white (​w) locus was identified by red (+) or white (​w) eye color, the

forked (​f) locus was identified by straight (+) or the presence of forked/bent (​f) bristles along the

posterior abdomen, and the ​miniature (​m) locus was identified by long wings past the abdomen

(+) or miniature wings that do not pass the abdomen (​m).

In the first week, the adults from the parental cross (+++ ​x​ ​wfm) were removed from their

tubes. They were anesthetized using carbon dioxide, categorized by sex and genotype with the

use of a dissecting microscope, then placed in a vial (containing 4 males and one female) to

mate. Since the P​1​ generation was already in the adult phase, they were given one week to breed

and produce offspring for the F​1​ generation. In the second week the F​1​ generation, consisting of

mutant males (X​wfm​;Y) and heterozygous wild type females (X​+++​;X​wfm​), was collected and

transferred into new vials with fresh ​Drosophila medium. They were anesthetized using ether,

categorized by sex and placed into four new vials, each containing five females and 3-5 males in

order to produce a large F​2​ population. These flies were given another week to mate. In the third

week, the F​1​ flies were cleared and the vial was checked for larvae. The larvae were then given

two weeks to develop into adults.

In the fifth week when the F​2​ flies were all adults, they were cleared from their vials,

anesthetized with ether, then phenotyped using a dissecting microscope. The F​1​ cross was to

produce offspring with 8 different phenotypes. Gene order and map distances were determined

using the X​2​ test, % of recombination, coefficient of coincidence, and interference.

Results

Table 1 shows the phenotype and number of flies in the F​1​ generation. It consisted of 16

mutant males (X​wfm​;Y) and 20 heterozygous wild type females (X​+++​;X​wfm​). The P​1​ generation

had consisted of four wild type males (X​+++​;Y) and one fully mutant female (X​wfm​;X​wfm​) which

was all that was needed to produce enough F​1​ offspring.

Table 1: Phenotypes and Number of Offspring in the F1 Generation

Phenotype Number of flies
Males X​wfm​
;Y 16
Females ​ ; Xwfm
X+++​ ​ 20

The table shows the expected phenotypes of the P​1​ cross. Females and males of the F​1​ generation

were able to be distinguished by eye color (red for females, white for males). Table 2 shows the

phenotypes and number of flies of the F​2​ generation for both males and females.

Table 2: Numbers and Phenotypes of F​2​ Offspring Resulting from the F​1​ Cross
Phenotype Number of males Number of females
+++ 160 157
wfm 28 57
+fm 17 27
w++ 60 82
++m 37 60
wf+ 10 15
+f+ 33 26
w+m 81 96
Total = 426 Total = 520

The greatest number of flies fell into the category of fully wild type for eye color, bristles, and

wings while the least number of flies were mutant for eye color and bristles and wild type for

wings. The sex of the flies is roughly a 4:5 ratio of males to females.
 Table 3 shows the X​2​ analysis for each of the traits in the F​2​ population by themselves.

The expected ratio for this analysis was that of a monohybrid testcross for each trait inherited

independently; each trait having a 50% chance of being inherited.

Table 3: Chi-Square Test of Independent Assortment for the Three Traits (wfm)
Trait 1 (wild-type vs. white eyes)
Phenotype Observed (O) Expected (E) (O-E)2​ O-E)2​​ /E
wild-type (red) 517 473 1936 4.09
mutant (white) 429 473 1936 4.09
Trait 2 (wild-type vs. forked bristles) Σ = X​ = 8.18
2​

Phenotype Observed (O) Expected (E) O-E)​ 2

O-E)2​​ /E
wild-type (straight) 733 473 67,600 142.9
mutant (forked) 213 473 67,600 142.9
Trait 3 (wild-type vs. miniature
wings) Σ = X2​​ = 284.2
Phenotype Observed (O) Expected (E) O-E)​2 O-E)​2​/E
wild-type (long) 543 473 4900 10.36
mutant (short) 403 473 4900 10.36
Σ = X​ = 20.72
2​

The trait with the highest deviation from the expected value was the forked (f) bristle trait. The

trait with the lowest deviation from expected value was the white (w) eye color trait. However,

all X​2​ values are very high. Table 4 similarly shows the X​2​ analysis for each different phenotype.

Tables 4: Chi-Square Test of Independent Assortment for each Phenotype

Phenotype Observed (O) Expected (E) (O-E)^2 (O-E)^2/E
+++ 317 118.25 39501.56 334.05
wfm 85 118.25 1105.56 9.35
+fm 44 118.25 5513.06 46.62
w++ 142 118.25 564.06 4.77
++m 97 118.25 451.56 3.82
wf+ 25 118.25 8695.56 73.54
+f+ 59 118.25 3510.56 29.69
w+m 177 118.25 3451.56 29.19
X2 = 531.02
The expected values were derived from a trihybrid testcross of the three traits assorting

independently; each having a 12.5% chance of being inherited. Some phenotypes are close to the

expected value, but the overall X​2​ value is extremely high.

Figure 1 shows the map distances of the three traits derived by calculating the chance of

recombination.

Figure 1: Genetic Map of Traits

w|​<--------------44.6 m.u.-------------->​m|​<---------37.8 m.u.--------->​|f

<----------------------------~70.4 m.u.----------------------------------->

These values were calculated based on the observed values for each of crossover. The map shows a greater chance of
recombination between ​w and ​f than between ​f and ​m.

The calculated coefficient of coincidence was calculated by dividing the observed double

cross-over frequency (12.9%) by the expected double cross-over (16.9%). This gave a value of

0.763. The interference is then calculated by subtracting the coefficient of coincidence from 1,

which yields a value of 0.237. The interference value is greater than zero. Therefore, positive

interference occurred, meaning that fewer double crossover events were observed than expected.
Discussion

Normally, traits on nonhomologous chromosomes are inherited independently of each

other’s locations. However, traits that are linked on homologous chromosomes are often

inherited together unless crossing over between the homologous chromosomes occur ((Klug et.

al. 2012)). The hypothesis for this experiment was that the three traits would be X-linked and not

inherited independently. Based on the results obtained from this experiment, this hypothesis can

be supported. At the same time, Mendel’s law of independent assortment would be rejected.

Referring to table 1 and the phenotypes of the P​1​ generation, the presence of fully wild type

males in the P​1​ generation disappeared in the F​1​ generation. This occurred because their X

chromosome was only inherited from the P​1​ female (which was fully mutant) and their Y

chromosome (which is without any of these traits and therefore uninfluential) was inherited from

the male. Meanwhile, F​1​ females inherited one fully wild type chromosome from the P​1​ males

and one fully mutant chromosome from the P​1​ female. If these genes were autosomal, then the

males of the F​1​ generation would be heterozygous wild types along with the females because it

would factor in an influential chromosome unlike the Y chromosome which contains none of the

three traits (Rodell et. al. 2004).

Table 2 shows the number and phenotypes of the F​2​ males and females. There are

significant differences between the numbers of offspring for each phenotype. If these traits had

assorted independently, then each phenotype would have about the same number of offspring (⅛

of total) because the probability of inheriting each of the three traits would be ½ , according to a

trihybrid testcross (Rodell et. al. 2004). Table 4 validates this with a Chi-Square analysis for

independent assortment for each phenotype. The calculation gives a X​2​ that is well above the
accepted X​2​ value of 14.067 (where df = 7 and p-value = 0.05) (Freund et. al. 2010). Table 3

shows a similar Chi-Square test, except it tests the independent assortment of each trait alone.

Once again, the X​2​ for each trait is above the accepted X​2​ values (Freund et. al. 2010). High X​2

values such as the ones shown in tables 3 and 4 are enough to reject independent assortment and

retain X-linkage.

Figure 1 displays the established genetic map, map distances and gene order of the traits.

The gene order, ​wmf, was determined by comparing the genotypes with the most offspring to the

ones to the least offspring and making sure they matched (Klug et. al. 2012). The data generally

correlates with the genetic map except it doesn’t match the single crossovers. For example, since

w_m is a larger distance than ​m_f, the observed recombination should be higher, however, it’s

lower. This could partially be due to mischaracterizing bristles on the flies because it is the trait

that is hardest to determine and easily mistakable. Also, some of the vials with the F​1​ cross did

not produce enough offspring, so more had to be taken from a previously prepared stock.

The gene order puts the gene for wing size (​m) in the middle of the other two traits. The

map distance between ​w and ​m is 44.6 map units, the distance between ​m and ​f is 37.8 m.u. and

the distance between ​w and ​f is 70.4 m.u. (which is slightly less than the addition of the first two

distances because the double crossover is counted twice) (Klug et. al. 2012). Ultimately, the data

obtained from this experiment generates the conclusion that the traits are X-linked and not

independently assorted.
References

Klug, W. S., Cummings, M. R., Spencer, C. A., and Palladino, M. A. (2012) ​Concepts of

Genetics (10th edition). San Francisco, CA: Pearson Benjamin Cummings, Inc.

Aggarwal, D. D., Rashkovetsky, E., Michalak, P., Cohen, I., Ronin, Y., Zhou, D., … Korol, A.

B. (2015). Experimental evolution of recombination and crossover interference in

Drosophila caused by directional selection for stress-related traits. ​BMC Biology, ​13,

101. doi: 10.1186/s12915-015-0206-5

Rodell, C. F., Schipper, M. R. and Keenan, D. K. (2004) Modes of Selection and Recombination

Response in ​Drosophila melanogaster.​ J Hered 95 (1): 70-75. doi:10.1093/jhered/esh016

Freund, R. J., Wilson, W. J., and Mohr, D.L. (2010) ​Statistical Methods (3rd edition).

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