A three-point test cross was conducted in Drosophila to determine the order and distance between three linked genes (y, cv, and f) on the X chromosome. F2 progeny were scored for phenotypes to determine the number of each genotype. Recombination frequencies and a genetic map were constructed based on the data. Chi-square tests compared the observed and expected values, and some deviations from expectations were observed. The results provided insight into genetic mapping and factors that influence mapping accuracy.

1. Recombination and Linkage
A Three point test cross in Drosophila
Linkage
Relationship between two or more genes near each other on the
same chromosome is known as Linkage
Law of independent assortment applies to genes on different
chromosomes(non linked genes)
Linkage
In a three point cross if there is no linkage what would be the
expected ratio for all classes ?
1:1:1:1:1:1:1:1
Aim for three point linkage experiment
Locate the respective position of the three linked gene loci on
the same chromosome
Measure distances between genes
Construct the genetic map which estimate the physical distances
between gene loci
Drosophila Genes & Phenotypes
Genotype (alleles)
y cv f mutant and + + + (Y CV F) wildtype
Phenotype (appearance)
Mutant: wild type:

2. y: yellow body grey body
cv: crossveinless wings crossvein wings
f: forked bristles straight bristles
Genes are located on the X chromosome
Parental Cross:
+++ / Y X ycvf / ycvf
Males Females
Phenotype (P): [WT] [yellow, crossveinless,
forked]
F1 Generation:
ycvf / Y and +++ / ycvf
Males Females
P: [yellow, crossveinless, forked] [WT]
F1 trihybrid test cross:
ycvf / Y X +++ / ycvf
4 Males 6 Females
First day lab set up
Test Cross-Testing genotype of parents showing dominant
phenotype
Test cross- Cross between F1 progeny and recessive parents.
F1 trihybrid test cross:
ycvf / Y x +++ / ycvf
4 Males 6 Females

3. During meiosis of spermatogenesis male Drosophila flies do not
exhibit crossing over between homologous pair - no
recombinant product.
X chromosome and Y chromosome do have regions of pairing,
and do form tetrads, but don’t recombine.
Male show complete linkage, advantageous for our experiment.
Tri-hybrid females had crossing over .
8 Phenotypes You will be expecting today!!
Grey, crossvein,straight (+ + + )
Yellow,crossveinless,forked(y cv f )
Grey,crossveinless,forked (+cv f )
Yellow,crossvein,straight(y + +)
Grey,crossvein,forked (+ + f)
Yellow,crossveinless,straight (y cv +)
Grey, crossveinless,straight(+cv +)
Yellow,crossvein,forked(y + f)
Place 10 -12 flies at a time to get the phenotypes.
Non-recombinant
Single CO- region I
Single CO-region II
Double CO
Our results
pool out the whole class data to calculate the genetic distance
more accurately

4. Today’s Goal: Construct a crude genetic map
Find the gene position
calculate c.o.c., interference
Perform X²(to determine if the map distance from the pooled
class data are statistically same with the published map
distance)
F2 Trihybrid Test Cross example
axp / +++ X axp / Y
gametes a x p YClass#F2+ + ++ + +/ a x p+ +
+/ Y162a x pa x p/ a x p a x p/ Y252+ x p+ x p/ a x p+ x p/
Y314a + +a + +/ a x pa + + / Y416+ + p+ + p/ a x p+ + p/ Y54a
x +a x +/ a x pa x +/ Y62+ x ++ x +/ a x p+ x +/ Y727a + pa +
p/ a x pa + p/ Y823

5. ** phenotypes of both sexes are similar, gender does not matter
F1 Parents-
F2 Data Analysis: Assigning
ClassesphenotypesClass#F2+ + +162a x p252+ x p314a + +416+
+ p54a x +62+ x +727a + p823
Non-recombinants (parental) usually have greatest #
Double crossover classes usually have smallest #
Single crossover I
Reciprocal classes have similar numbers
Single crossover II
F2 Data Analysis: 1st goal- determine the order of loci
We will be comparing P & DCO classesphenotypesClass#F2+ +
+162a x p252+ x p314a + +416+ + p54a x +62+ x +727a + p823
200

6. + + +
a x p
+ + p
a x +
a+ p x+
a p+ x
a+ p+ x+
a p x
In DC classes ,middle gene gets inverted from its initial
position in the parental chromosome
F2 Data Analysis: Rearrange and Calculate Recombination
percentage
RF = phenotypesClass#F2+ + +162a p x252+ p
x314a + +416+ + x527a p +623+ p +74a + x82

7. 200
RF I = = 0.18
Between a and p
RF II = = 0.28
ap units
Between p and x
Note that F2# is greater for CO between p and x
F2 Data Analysis: Drawing the Map based on recombinant
frequency
1 map unit = 1% recombination
28 map units in Region II (p and x)
a p x
18 m.u.

8. 28 m.u.
2nd step: Calculate C.O.C and interference
Coefficient of coincidence(c.o.c.) =
Expected DCO = (RF I)(RF II)(Total #)
Interference(I) = 1 – c.o.c
Exp DCO = (0.18)(0.28)(200) = 10.08
c.o.c. = = 0.595
I = 1 – 0.595 = 0.405
~ 40% interference
terference
Negative value means scoring is wrong
C.O.C-measure the amount the interference of one CO to reduce
the formation of another CO
Third Goal:X² Analyses ( Observed vs published distance
between 1st and 2nd gene)
rved and accepted are due
to….
a
p
x
13.7 mu
43 mu X² for Region IObservedExpected(f-f*)2/f*Recomb.
I14+16+4+2=

9. 36(RF1 Pub) x total 0.137x200=27.42.7NonRecomb ITotal-
recomb
200-36=164Total-recomb
200-27.4=172.6 0.43
X2 = 3.13
df = 1
X² Analyses: Observed vs Published for Region II
due to….
a
p
x
13.7 mu
43 mu
X2 = 18.4
df = 1X² for Region IIObservedExpected(f-f*)2/f*Recomb.
II27+23+4+2=
56(RF2 pub) x total 0.43x200=8610.5NonRecomb IITotal-
recomb
200-56=144Total-recomb
200-86=1147.9
X² Analyses: Reciprocal Classes
Numbers for reciprocal classes should be 1:1 ratio
X² Class 1&2ObservedExpected(f-f*)2/f*Class 162½ (total) =
½(114)
570.44Class 252½ (total) = ½ (114) 57 0.44
X2 = 0.88
df = 1

10. chance
Possible Sources of Error
Over-estimation of RF
Inadequate sample size
Misscoring of flies
Missing double crossovers
Under-estimation of RF
Some crossover flies are less viable and selected against
Ex. Triple mutants less viable than double/single mutants
Failure to detect multiple crossovers in a given region
**Do not want to hear the typical “standard experimental error”
Ways to Improve
Score phenotypes more carefully.
It is better to use more markers so RF is not being measured for
distant genes that would have lots of crossovers, such as cv-f
region, unlike the y-cv region, which is smaller, so less DCO
occur.
Increase sample size
In summary
1. Determine the order of the three genes on the chromosome
and find the middle gene
2. Calculate the recombination percentages for the two
intervening regions

11. 3. Construct the genetic map
4. Calculate the COC and Interference
Genotype
Observed
Type of Gamete
Y F CV
310
Parental
y f cv
260
Parental
Y f cv
70
Single-crossover I
Y F CV
80
Single-crossover I
Y F cv
20

12. Double-crossover
y f CV
10
Double-crossover
Y f CV
135
Single-crossover II
y F cv
115
Single-crossover II
Total
1000
Following is the published distances for three genes
1st
middle
3rd
13.7 mu
43 mu

13. Make up Data_Sec 06
Drosophila Three-Point Test Cross Lab Write-Up
Instructions(65 points)
Abstract (5 points)
In a short paragraph describe the experiment that was done as
well as the major findings. Clarity is essential. The abstract is
usually written last and is limited to 200 words.
Introduction (14 points)
Provide ALL background information a reader would need to
understand the purpose, results and analysis of the experiment.
Must include:
1. Why it is important to know the locations of genes in the
genome.
2. A description a crossing-over during meiosis, linkage,
recombination frequency (RF), and how RF relates to map
units? How does RF change for closely linked versus distantly
linked and unlinked, genes?

14. 3. Why is it advantageous to map three (or more) genes at once
instead of mapping each pair of genes separately
4. The benefits of Drosophila for genetic analyses
5. The hypotheses for this experiment
· RF measured in lab will be similar to the expected RF based
on known map distances.
· Reciprocal classes will occur and survive in equal numbers.
· Interference will be a positive value.
Methods (14 points)
This section should provide enough information so that the
reader could carry out the experiment independently.
1. Explain the experimental strategy: P, F1 and F2. Describe all
genotypes and expected phenotypes.
2. Describe the different traits that were scored.
3. Why was it unnecessary to determine the sex of the F2?
4. Describe calculations for RF, map units, and Interference.
5. Describe the Chi-Square tests that were done and the highest
acceptable Chi-Square value for a corresponding p value of 0.05
or lower for relevant degrees of freedom used in your different
Chi-Square tests. (Measured vs. published map distances;
reciprocal classes)
Results (14 points)
In this section, the data are shown in tables AND explained in
coherent paragraphs.
1. Produce a table with the counts of each F2 phenotype for:
your group’s data, your lab section’s data, data provided by the
fly experts. (Note to TA’s: This lab has a long history of
terrible data, so each lab instructor will invent a dataset for
each of her/his lab sections. These data sets MUST change each
semester!)
2. Produce THREE genetic maps, each based on each of the data
sets in the Table. Calculate Interference for each data set. Show

15. the equation for calculation of interference.
3. Compare expected and observed data for pairwise map
distances among the three genes and for reciprocal crosses using
Chi-Square values. Report p-values for all comparisons, and
state whether differences between expected and observed data
can be attributed to chance. Do this for all data sets. (There will
be 18 Chi-square calculations, 6 per data set.)
4. A narrative must describe the table, mapping calculations and
Chi-Square calculations. You must interpret your Chi-Square
results. Can deviations from expected values be attributed to
chance? Explain your reasoning.
Discussion (14 points)
The results are summarized in this section and the reasons WHY
data were significantly different than expected are considered.
1. How do map units calculated from the three data sets (one
small and two large) compare to published distances?
a. What happened for the shorter y-cv distance?
b. What happened for the longer cv-f distance?
c. What happened with the 4 reciprocal classes? In the case of
reciprocal classes, were any trends observed (certain reciprocals
tend to be near equal while others were quite different)? How
do mutations affect viability?
d. Did these results match the hypotheses stated earlier?
2. Why is it difficult to accurately measure long map distances
by RF?
a. What can be done for more accurate measurements of long
map distances?
3. What difficulties arose when assigning phenotypes when
scoring the F2?
a. What could be done to reduce these difficulties?
Overall Conclusions (4 points)
Keep this section short, one paragraph at the most. Do not
repeat yourself over and over when writing this paragraph!
What do the data demonstrate?

16. Why is a statistical analysis important?
Summarize ways to improve the outcome of the three point
testcross mapping experiment; describe “tricks” for evaluating
phenotypes.
PLAGIARISM: Remember, you must use your own words, even
if you work with others to discuss what the content of your
paper will be. Do not use quotations; read material, figure out
what it means, and then explain in your own words. If you do
use material not found in the lab manual or the textbook, be
sure to cite it. Instructions for citations are found in the oral
presentation section of the Genetics Lab Manual. All papers
must be .doc or .docx files, and will be submitted to your lab’s
BeachBoard Dropbox and will be subject to plagiarism detection
using Turnitin. A strict ZERO policy (on the entire write-up)
will apply to all plagiarism that goes beyond a shared, common
phrase. If two students’ papers are found to be highly similar,
BOTH students will receive a ZERO. Do not give your word
file to a friend to help them out at the last minute; they will
likely take both of you down. Papers must be uploaded to the
lab BeachBoard Dropbox BEFORE your lab starts on the
designated due date. Please see
http://philosophy.tamu.edu/~gary/intro/plagiarism.index.html
for some examples of plagiarism.
Citations
To respond to question 1 of the Introduction, you will need to
look up papers. Cite these as described in the group oral
presentation instructions in the Genetics Lab Manual.
Writing Tips
Many students feel that if they write something in complicated
language, they sound more intelligent. This results in awful
sentences such as, “A significant frequency of DNA is made of
gene.” “Genes are made of DNA.” makes a lot more sense!
Also, the term “significant” is only used with an accompanying
statistical test. See below for more helpful writing tips:

17. 1) The phrasing, ", so..." is conversational English, and not
appropriate for written English.
2) The word "very" has little meaning. Use a stronger
adjective. Four letter V-WORD.
3) Use the passive voice, not "We define recombination
frequency as..." Instead use: "Recombination frequency is
defined as..."
4) Separate different sections into paragraphs so the overall
organization is clear to the reader.
5) If you want to use "it" or "they" in a sentence, be certain that
the subject referred to is clear.
6) Omit needless words. Go through each sentence to reduce
wordiness.
7)” it’s” = it is; “its” is the possessive.
8) Do not keep using the word “it” in your complex sentences.
Re-word the sentence so the subject is clear.
9) Avoid meaningless sentences such as “Chromosomes are
interesting molecules that are found in Drosophila.” Think of a
real point you want to make, and use meaningful language.
10) Avoid contractions; don’t use them! I cannot emphasize
this enough; they’re too informal.
11) Semicolons separate two independent clauses; independent
clauses can serve as their own sentence.
12) A colon separates one independent and one dependent
clause: as in this sentence.

18. 13) The possessive is rarely used in scientific writing and
comes off as awkward and unprofessional. Do not write, “The
gene’s location is not known.” Instead, write, “The location of
the gene is not known.”