Download Activity 1: F1 Drosophila. Your group will receive two vials of

LABORATORY SESSION 8: TESTING GENETIC HYPOTHESES PART 1.
Activity 1: F1 Drosophila.
Your group will receive two vials of Drosophila melanogaster. These flies (unmated
males and females) are the F1 generation of a cross set up by the lab coordinator almost
two weeks previously. A complete list of all the crosses is given at the end of this
activity. Each cross involves mutants at two different loci.
You will also receive vials containing samples of the parental strains. Compare the
phenotypes of parents and offspring. Note which traits appear to be recessive and which
appear to be dominant. Also note whether there seems to be any sex linkage (depending
on the cross you may not be able to tell).
Come up with a specific hypothesis for the genotypes of the parents and the F1 flies. Use
this hypothesis to predict what the F2 offspring should look like (i.e. what phenotypic
ratios you expect). Also what the results of a test cross would look like.
An example. The parents were apterous sepia females (having no wings and dark brown
eyes) and wild type (non-mutant) males (red eyes and wings). All the F1 have the wildtype phenotype for both wings and eyes.
Your hypothesis – apterous (allele – ap) and sepia (se) are both recessive to the normal
(wildtype) alleles at their loci (wildtype is designated by a + sign)
Parental females were homozygous recessive at both loci (ap/ap, se/se) while the males
were homozygous for the wildtype allele at both loci (+/+, +/+).
The F1 were double heterozygotes (+/ap, +/se).
If both loci are autosomal (not on sex chromosomes) and not linked then the expected
phenotypic ratio is the standard 9:3:3:1 (ask your TA to explain this if your memory is
hazy).
You would expect 9/16 wildtype both traits, 3/16 apterous with wildtype eyes, 3/16 sepia
with wildtype wings, and 1/16 apterous and sepia.
Prepare a similar prediction for your flies. Set up the crosses.
Where possible (i.e. where we have the appropriate flies) also set up a test cross –
crossing the F1 flies to flies homozygous recessive for both traits.
In traits where you expect sex linkage think about the kinds of crosses you might want to
make. Ask your TA if you need help.
Present your hypothesis and how you intend to test it to the class.
Table of Potential Crosses
Female
Sepia (eye color) and ebony (body color)
Dumpy (wing shape) and sepia (eye color)
Yellow (body color), white (eye color)
White (eye color), wildtype (wing)
Brown (eye color)
Brown (eye color), wildtype wing
Brown (eye color) wildtype antennae
Antennapedia (antennae like legs),
wildtype eyes
Antennapedia (antennae like legs),
wildtype eyes
Antennapedia (antennae like legs),
wildtype wings
Vestigial (wing), wildtype eye
Vestigial (wing), wildtype eye
Vestigial (wing), wildtype antennae
Male
Wildtype for both traits (red eyes, brown
body)
Wildtype for both traits (red eyes, brown
body)
Wildtype for both traits (red eyes, brown
body)
Wildtype (eye color), vestigial (wing)
White eye color (different locus from
brown)
Vestigial (wing), wildtype eye
Antennapedia (antennae like legs),
wildtype eyes
White (eye color), wildtype antennae
Brown (eye color) wildtype antennae
Vestigial (wing), wildtype antennae
Brown (eye color), wildtype wing
White (eye color), wildtype (wing)
Antennapedia (antennae like legs),
wildtype wings
ACTIVITY 2: EPISTASIS
The fruit (ears or cobs) of corn (maize) can be used to study genetic ratios. Each kernel
is one offspring. For traits that can be scored in the seeds a large number of offspring can
be scored very easily.
Observe the ears of corn labeled for epistasis cross 1. The ears are the result of the cross
of a double heterozygote. The two gene loci in question are not linked, but one does
influence the expressin of the other. One gene (R) is a dominant gene that is responsible
for the production of a red pigment in the corn kernels. The second dominant gene (C')
inhibits the action of the R gene locus.
Count a sample of kernels from the ear and determine the phenotypic ratios of the
offspring. Use that information to develop hypotheses relating the genetic makeup of the
parents to the genotypes and phenotypes of the offspring. What are the possible offspring
genotypes and what phenotype does each genotype produce.
Calculate chi square probabilities to check your hypotheses using the information in your
lab manual.
Record your data, hypotheses, and chi square probability in your notebook.
Now move on to the ears of corn marked epistasis cross 2. These parents of these ears
were homozygous for the R allele. However they were double heterozygotes for C1
locus and a third locus, the Y locus. Count a sample of kernels as you did before (note
that there are now three different colors) and repeat the process you followed for the first
ear. How do the two loci interact to produce color in this example?
ACTIVITY 3: GENETICS AND COMPLEX LIFE CYCLES (Ferns have not developed –
retain for possible future reference after spring break).
All plants have two different multicellular stages in their life cycle: a gametophyte
(haploid) and a sporophyte (diploid). In flowering plants the gametophyte consists of
only a handful of cells. However in ferns and other seedless plants both stages have an
independent existence. Fern sporophytes are the comparatively large leafy plants that we
are familiar with. Fern gametophytes are typically flat green organisms less than 1 cm
across that occur on the surface of the soil (some are non-photosynthetic and live
underground).
C-Ferns are ferns that, much like the fast plants, have been bred to grow well and quickly
in the lab. The gametophytes are growing on agar plates. Each gametophyte is a flat
green structure. On top of the gametophyte are the gametangia – the structures that
produce gametes. Unlike most seed plants, ferns have motile sperm, capable of
movement like animal sperm. Sperm are produced in antheridia (male gametangia) while
eggs are produced in archegonia (female gametangia). Look at the diagrams of
gametophytes in the lab to familiarize yourself with these structures.
We will supply you with plates with four different groups of gametophytes. If the
cultures are advanced enough we may also see young sporophytes.
Single gene mutants. Look at the plates labeled Bubbles and Highly Male. Highly male
is a mutation that causes the gametophytes to produce very high numbers of antheridia.
Bubbles causes swelling of some cells in the gametophyte.
Each of these is growing in pure culture (all gametophytes have the same genotype).
What genotype(s) could a sporophyte have that is produced from the highly male culture?
What do you think such a sporophyte would look like? Consider the same questions
about the Bubbles culture?
F1 Stocks (monhybrid and dihybrid)
Polka dot – This mutation has reduced chlorophyll and produces pale cells with dark dots
in them. A polka dot (recessive) gametophyte was crossed to a wildtype (dominant)
gametophyte. What do you think the sporophyte looked like? Why?
What types of gametophytes do you see? In what ratio? Assuming the gametophytes
mate randomly what genotypes of sporophytes would you expect these gametophytes to
produce? In what ratio?
Polka dot and dwarf. The sporophyte that produced these gametophytes is a double
heterozygote (DWF1/dwf1, CP/cp) where the normal phenotype is produced by the
dominant allele in both cases. What types of gametophytes do you expect to see? In
what ratio? What types do you see and do the results match your expectation? What
types of sporophytes do you expect to see? What do you see?
ACTIVITY 4 - SCORING THE GENES AFFECTING COAT COLOR IN DOMESTIC CATS
(outside of class exercise)
Each student will gather data on at least five cats. You can use your own pet cats, the
pets of friends or family, cats you observe on the street, or you can take a field trip to the
humane society or other shelter. Ideally you should record the sex of each cat but this
may not be possible for every individual. You should also record the type of cat (pet,
stray, etc.) and the geographic location (Champaign-Urbana area, elsewhere in Illinois) in
as much detail as you can.
Record data on four loci: agouti, tabby, orange, spotting.
You will record data on phenotype (and where possible genotype) at four loci controlling
coat color in domestic cats. All these alleles control the deposition of melanins
(pigments) on the hairs. There are numerous other loci also affecting coat color that we
will not be concerned with but that will be mentioned briefly at the end.
The Agouti Locus – This locus controls the pattern of melanin deposition on individual
hairs. Cats with the genotypes AA and Aa have agouti fur meaning that each hair is
banded with a dark tip, lighter middle and darker base. Hairs with the agouti pattern
produce fur that is ticked, i.e. not solid in color but with a grizzled appearance. Cats that
are aa have solid colored fur (the actual color depends on genes at other loci).
Agouti has complete dominance – you will not be able to distinguish AA and Aa cats.
Record them as A-. Do not record a genotype for the agouti locus for any orange cats,
orange coloration masks agouti.
The Tabby Locus – The tabby locus controls the pattern of solid versus agouti fur. Cats
that are aa at the agouti locus do not express the tabby phenotype. In other words if a cat
is aa, its genotype at the tabby locus has no effect on its color pattern because all the fur
is solid and there is no agouti fur. Therefore you will only be able to score this locus if
the cat is A-. Tabby has three alleles: T, Ta, and tb. TT and Ttb produce cats with the
common striped, tabby pattern (called Mackerel Tabby by cat breeders). The
homozygote tbtb produces the so-called classic tabby in which the solid color is mostly in
blotches and swirls rather than stripes. The Ta allele produces the so-called Abyssinian
coloration in which all the fur is agouti with no solid patches or stripes. TaTa cats have
no solid markings, TaT and Tatb cats will have some striping on the legs and tail.
The Orange Locus – This is an X-linked allele. The O allele causes orange coloration
that masks the agouti allele. All orange cats show their tabby genotype. Males are either
OY (orange) or oY (not orange – color determined by other loci). Females can be OO
(orange – rare), oo (not orange) or Oo (calico or tortoiseshell). The blotched pattern of
calico and tortoiseshell cats results from the inactivation of one X chromosome or the
other in different cells early in development.
The Spotting Locus – This locus prevents melanin from being deposited on hair,
resulting in white fur. White spotting shows incomplete dominance to normal coloration.
Cats with genotype ss have no patches of white fur. Cats with SS or Ss have some white
fur and some fur with other coloration. SS cats generally have more than half their fur
white, Ss cats usually have less than half white fur.
Scoring the S locus will involve estimation and guess work on your part and thus a
certain amount of experimental error. Score all cats with <50% white as Ss and >50%
white as SS. Some SS cats are completely white but do not score them. Pure white cats
can be produced by at least two other loci.
Other loci – There are several other loci such as Dilute and Black, which have alleles
that modify the basic color (turning black fur into blue or brown fur for example). Most
of the recessive alleles for these loci are rare in feral and pet cats. We will not score these
loci. These alleles should not interfere with your scoring the Agouti, Tabby, and Spotting
loci. Rare loci at some loci may make it difficult for the inexperienced to score orange.
If you encounter a cat with unusual pale brown/buffy coloration you should probably not
score it at the orange locus. This is not very likely.
Cats you shouldn’t score. Do not score any pure white cats. This can be caused by
alleles found at three different loci. In all cases it is impossible to determine the genotype
of white cats at other loci just from their appearance. Also do not score any Siamese or
Burmese type cats. The color patterns of these cats are caused by two alleles at the Color
Extension locus. Although it is possible to score these cats for the loci described above,
they have very different allele frequencies at some loci. In particular the A allele and the
S allele (very common in cats of European ancestry) are almost completely absent in
these cats. Also do not score any registered breed cats (i.e. cats produced by professional
breeders that could be shown in cat shows). These cats have been bred for particular
characteristics and will likely have many rare alleles that could complicate scoring.
This may sound complicated. But if you use the following procedure scoring each cat
should not be difficult. When you find a cat go through the following steps in order from
1 to 5.
Step 1 – Should you score the cat? If it is pure white, Siamese/Burmese, or a pure bred
‘show’ cat – do not score it.
Step 2 - Spotting Locus – Estimate the proportion of the cat that is white. None = s/s,
Up to 50% = S/s. More than 50% = S/S. Tortoiseshell and Calico cats are O/o cats
without and with white spotting respectively. Estimate their S genotypes just like any
other cat.
Step 3 – Orange Locus – If the cat is all orange or orange and white it is O/O (if female)
or O/Y if male. If sex is unknown score as O/-. Calico or tortoiseshell cats are O/o. All
other cats are o/o or o/Y (unknown sex o/-).
Step 4 – Agouti locus. Cats that are all orange or orange and white cannot be scored for
agouti. Put a question mark in the A locus column for these cats. Cats with solid color
(non-white) are a/a, all others are A/- (to score Tortoiseshells and Calicos look at the nonorange (and non-white) patches of fur).
Step 5 – Tabby Locus. If the cat a/a and o/- (steps 3 and 4), then you cannot score this
locus (put a question mark in the column). If it has solid stripes on agouti or orange fur it
is T/T or T/tb. If it has blotches and/or swirls then it is tb/tb. If it has no striping or only a
little on the legs and tail then it is Ta/-.
Note: In some cats with extensive white spotting and in some Tortoiseshell cats it may
be very difficult to score the T locus because the spots of agouti or orange fur are too
small to see the pattern. If you cannot score the locus after an honest effort then record a
question mark.
Here are two web sites with links to cat coat color genetics. Your assignment (due in
three weeks) is to find 5 cats and genotype as described in the lab manual. These can be
cats you own or who belong to friends or family of yours. Or they can be cats you see in
the street or elsewhere.
www.janecky.com/runyen/bio554/cat/INDEX.htm - this is a slide show with images of
the different kinds of cats - you may be shown one of these images on a quiz
www.fanciers.com/other-faqs/color-genetics.html
Record your data in the following table. It has eight columns. Give each cat an
identifying number in the first column (1to10). Record the date and time in the second
column, the location in the third. Record the genotype for the appropriate locus in
columns 4-7. Record a verbal description of each cats coat on a separate page and any
additional comments in column 8
Numb
er
Date/
Time
Location
S locus
O locus
A locus
T locus
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