BISC413 Lab 1, Sept. 1: Flies and cats

Transfer fly population to new vial

Your first task today will be to begin your semester-long project on a visible mutation in Drosophila melanogaster. You will be handed a vial containing a mixture of wild-type and mutant flies. On August 18 I took adult flies from pure lines (homozygotes) of wild-type and mutant flies. I put some mutant flies (about 10 to 30; I didn't count them) from one line in each vial, along with a smaller number (about 5 to 10) of wild-type flies. On Aug. 27 I removed the adults, leaving the larvae and pupae in the vial. Today you should have plenty of adults. The flies I used to set up the vials were not virgins, so most of the offspring you see are probably the result of wild×wild or mutant×mutant matings. There are 8 different mutations in the class; each mutant has two people working on it.

Part of your semester-long project will be to follow the allele frequencies over several generations. Ideally, you'll transfer the adults to a new vial of food every other Tuesday, then on the following Thursday remove the adults, anesthetize them, and count the number of mutant and wild-type flies. If everything works, you should have six generations of data by the end of the semester. If there is strong directional selection, you should see the allele frequencies going up or down over that time.

Next week you'll start to plan an individual project to investigate your mutant in more detail. Today you'll just make a new vial of food and transfer your adults to it.

  1. Record the information on the label on your vial.
  2. Label a new vial with the symbol for your mutant allele, the date, and your last name. Always write on tape, not directly on the vial, so the vials can be reused.
  3. Put one level teaspoon (5 ml) of dry fly food into the vial. Then add one teaspoon of distilled water. Jiggle the vial back and forth so that the water mixes in with the food; it should look like thick soup when you jiggle it.
  4. Add a few grains of dry yeast.
  5. Put a foam plug in the vial, so stray flies don't get in it, and let the food set for a few minutes.
  6. Remove the foam plug and insert a funnel.
  7. The next part requires speed and coordination to keep flies from escaping, flying out into the halls, and annoying non-geneticists. You should probably get your lab partner to help you the first couple times you do this. First, knock the bottom of the old vial onto the lab bench a few times. This will knock the flies to the bottom of the vial. Then remove the foam plug, and put the vial upside-down into the funnel. You only have a couple of seconds to do this before the flies recover from being knocked down and start flying around.
  8. Once the vial is in the funnel, hold both the bottom and the top vials and knock the whole assemblage on the lab bench a few times. The goal is to get the flies knocked down through the funnel, into the new vial. Try to get all the adults into the new vial, but keep an eye on the food in the old vial; if it starts to slide down, stop.
  9. Once you have all the adult flies in the new vial (or as many as you can get without knocking the food down), knock the flies in the new vial to the bottom, remove the funnel, and put a foam plug in it. Put the foam plug back in the old vial.
  10. Put a rubber band around the old and new vials, and put them in the big tray of flies. Record anything unusual, like the food falling down, half the flies escaping, etc.

Make a fly trap

There are hundreds of scientific papers about a polymorphism in the alcohol dehydrogenase (Adh) gene in natural populations of D. melanogaster. There are two common alleles, called AdhFast and AdhSlow for the relative positions of their protein products on gels. Studies that have looked at large-scale geographic patterns have consistently found latitudinal clines—the AdhFast allele is rare in tropical areas and gets more common at higher latitudes.

A typical study of geographic patterns in allele frequency looks at one sample every few hundred kilometers, over a broad geographic range. We are going to see if there is significant variation in allele frequency on a smaller scale. You'll make a fly trap and put it somewhere in Newark. We'll discuss where to put them so that we have 8 different locations in Newark, with two people's traps in each location. On Thursday you'll bring the flies you've caught to lab, remove them from the traps, and freeze them. In coming weeks you'll run starch gels on the flies to determine their genotypes at the Adh locus.

Fly trap
  1. Select three plastic soda or water bottles of the same brand and size. Cut them with a razor blade as shown in the diagram.
  2. Mix 1/4 cup of mashed banana, three teaspoons (15 ml) of beer, a few grains of dry yeast, and one level teaspoon of corn starch. Put this in the bottom of bottle number 1.
  3. Put a piece of mesh over the bottom of bottle number 1, and hold it in place with a rubber band near the bottom of the bottle.
  4. Jam the top of bottle number 1, the one with holes in it, onto the bottom of bottle number 1. Pull the mesh up over the holes and hold it in place with the rubber band. Tape the two halves together.
  5. Jam the top of bottle number 2 onto the top of bottle number 1. Tape it in place.
  6. Jam the top of bottle number 3 onto the top of bottle number 2. Tape it in place.
  7. Make sure no flies from the lab got into your trap. Put a foam plug in the top bottle.
  8. Label the trap with your name. If you will be putting it in a public place, also label it "University of Delaware genetics experiment--do not disturb. For questions call" and your phone number.
  9. After class, take the trap to the place where you'll set it up. Put it someplace where it is out of rain or direct sunlight, preferably someplace where no one is likely to see it. Take detailed notes about where you put it. Put it there Tuesday evening and collect it sometime Thursday.

Cat coat genetics

In this lab, you and your lab partner will estimate the allele frequencies at four genetic loci in domestic cats, Felis catus (also sometimes called Felis domesticus, Felis sylvestris catus, or Felis sylvestris domesticus). You will collect data by looking at pictures of cats up for adoption in animal shelters. For two loci, you will estimate allele frequencies from phenotype frequencies using the Hardy-Weinberg relationship; for two loci, you will test the fit of the genotype frequencies to those expected under Hardy-Weinberg. You will then compare allele frequencies at two different geographic regions. Finally, you will think about possible problems with your sample and better ways of obtaining a more random sample of cats.

The goals of this lab are to reinforce basic concepts in Mendelian genetics (dominant/recessive, codominance, locus, allele), population genetics (Hardy-Weinberg), population sampling, and statistics. For those of you who will become teachers, variations on this lab are a good introduction to genetics for students ranging from third grade ("Are there more long-haired cats in colder areas?") to grad school ("What is the molecular basis of variable expression of piebald spotting?").

Domestic cats have several characters that are inherited in a simple Mendelian fashion; each is largely determined by one locus with two alleles. Cat breeders have worked out the inheritance of each character, deterimining which loci were autosomal and which were sex-linked, which alleles were dominant and recessive. This makes it easy to estimate allele frequencies by merely looking at a sample of cats; no breeding studies or molecular tests are necessary. For this reason, underfunded population geneticists in many parts of the world have surveyed allele frequencies in local populations of cats and have used the data to infer the processes of selection, migration and drift that have influenced them.

In this lab, you and your lab partner will look at pictures of cats up for adoption in two areas, one colder and one warmer. You will determine the phenotype of each cat, then estimate the allele frequencies in the two areas. For the orange and spotting loci, which are codominant, you will be able to count the allele frequencies directly; you can therefore test the fit of the genotype frequencies to Hardy-Weinberg proportions, using the chi-square test of goodness-of-fit. You will use the chi-square test of independence to compare allele frequencies between the two locations.

Procedure

You and your lab partner will be assigned one of the following pairs of locations, one warm and one cool. For United States and Canada locations, go to http://www.petfinder.com, choose "Cats," enter the city, click on the "Pic Preview" box, and click "Go." You should get a list of cats for adoption in and near the city you entered. Click on each thumbnail picture to get more details on each cat. There may be a magnifying glass icon at the lower right of a picture, to give you a better look. For foreign locations, go to the web address I've listed here.

 

warm: Queensland, Australia http://www.awlqld.com.au/rehoming.htm
http://www.maryboroughanimalrefuge.com/
cool: New South Wales, Australia: http://www.dchanimaladoptions.com/

 

warm: Israel http://www.israelpets.org/eng/
cool: Norway http://www.dooa.no/omplassering/katter.asp

 

warm: San Juan, PR
cool: Halifax, NS

 

warm: Miami, FL
cool: Portland, ME

 

warm: Mobile, AL
cool: Duluth, MN

 

warm: Houston, TX
cool: Winnipeg, MB

 

warm: Honolulu, HI
cool: Anchorage, AK

 

warm: San Diego, CA
cool: Seattle, WA

 

warm: Tucson, AZ
cool: Edmonton, AB

Look at the pictures and descriptions of as many cats as you can from each location. I'd like you to get information for 25 cats from each location, but some places don't have that many cats. You can use nearby locations to increase your sample size (for example, Vieqes, Mayaguez, and San Juan, Puerto Rico), but don't go too far away.

I suggest that one of you score the cats while the other writes down the information; switch roles when you switch locations.

For each cat, record the following information:

Name: So you don't score the same cat twice.

Sex.

Cat with short hair Cat with long hair
Atreyu, on the top, has short hair (genotype LL or Ll); Beauty, on the bottom, has long hair (genotype ll).

Hair length: long or short. (Some web sites describe some cats as having "medium" hair; include that with long, unless it's obvious from the picture that the cat really has short hair.) Hair length is controlled by the longhair locus, with the alleles L and l. ll cats have long hair, while Ll and LL cats have short hair.

Cat with white hair Cat with colored hair
Swayze, on the left, has all white hair (genotype WW or Ww); Myuu, on the right, has some colored hair (genotype ww).

White or colored: Cats with the WW or Ww genotype at the white locus have completely white fur; cats with the ww genotype have some color on them. Only count a cat as white if it is completely white; in some ww cats, the white patch caused by the spotting gene extends so far that there's only a little patch of color on top of the cat's head. If a cat has the W allele, you can't tell what genotype it has at the spotting, orange, agouti, or color loci.

Cat with more than half white hair Cat with some white hair Cat with no white hair
Tina T, on the upper left, has more than half of its body covered with white hair (genotype SS); Joe Dirt, on the upper right, has some white, but less than half (genotype Ss); Angel Poo, on the lower left, has no white hair (genotype ss).

Presence and amount of white spotting: Record whether each cat has some white patches on it, or is completely colored. If there is some white, estimate whether the white fur covers more or less than half the body. Cats with the ss genotype at the spotting locus have no white fur, while the Ss and SS genotypes have white patches. The extent of white fur in Ss and SS cats is variable, being determined by other genes and by environmental factors. Some sources say that cats with the SS genotype have white fur on more than half their body, while cats with the Ss genotype have white on less than half their body. I'm not sure whether we'll always be able to tell the difference between SS and Ss cats, and we may end up lumping them together.

Cat with orange hair Cat with orange and black hair Cat with no orange hair
Bitsy, on the upper left, has orange hair (genotype OO, since she's female; if this were a male, he'd be OY); Enid, on the upper right, has orange and black hair ("calico" or "tortoiseshell", genotype Oo); Bert, on the lower left, has no orange hair (genotype oY; if this were a female, she'd be oo). Note that the brownish hair on Bert's belly doesn't count as orange.

Orange/cream color present or not: The orange locus is on the X chromosome, so males are either OY or oY. An OY male is orange or cream colored, while an oY male is black, brown or gray. The darkness of the color (orange vs. cream or black vs. brown vs. gray) is determined by other genes that we won't try to score. An OO female is orange or cream colored, while an oo female is black, brown or gray. In an Oo female, one allele is inactivated in each cell early in development. Cells descended from a cell in which the O allele was inactivated will make black fur, while cells with the o allele inactivated will make orange fur. The result is a cat with patches of orange and black (or cream and gray) fur. This is generally called a "tortoiseshell" if there are no white patches and a "calico" if there are white patches.

Unknown: Some cats will be unknown for some characters, either because the picture is unclear or because the character cannot be scored in that cat. For example, spotting, orange, tabby, and siamese cannot be scored in all-white cats. And none of the characters can be scored in cats like the Colonel:

Cat with no hair
The Colonel.

Data analysis

If there's time, you can begin to analyze the cat data, as described in Thursday's lab.

Return to the Genetics Lab syllabus

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This page was last revised August 29, 2009. Its URL is http://udel.edu/~mcdonald/geneticslab1.html