Download Lab I: Three-Point Mapping in ​Drosophila melanogaster

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
Males
Females
X​
Number of flies
wfm​
16
X+++​
​ ; Xwfm
​
20
;Y
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)
(O-E)2​
Expected (E)
O-E)2​​ /E
wild-type (red)
517
473
1936
mutant (white)
429
473
1936
4.09
Σ = X​ = 8.18
2​
Trait 2 (wild-type vs. forked bristles)
Phenotype
4.09
Observed (O)
O-E)​
O-E)2​​ /E
2
Expected (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)
Phenotype
Observed (O)
Σ = X2​​ = 284.2
O-E)​2
Expected (E)
O-E)​2​/E
wild-type (long)
543
473
4900
mutant (short)
403
473
4900
10.36
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).
Burlington, MA: Elsevier, Inc.