Lab 7 – Heredity

Is there a fly in here?

Note: This lab is another “old lab” from the previous manual, however you’ll need to write it into your

lab notebooks as an informal report (not a packet).

The Background

• Drosophila melanogaster, also known as a fruit fly (or gnat), is a very common model for genetic studies.

• Its entire genome has been sequenced and thus we can create entire lineages of Drosophila by manipulating their genes.

• We’re going to explore this concept further by having you cross fruit flies with particular traits.

• Here are some common ones that have been made over the years through genetic modification.

Phenotypes

http://www.unc.edu/depts/our/hhmi/hhmi-ft_learning_modules/fruitflymodule/phenotypes.html

Wild Type Vestigial Winged

Curly Winged

YellowEbony

Antennapedia(head-legs)

Orange Eyed

White Eyed

Eyeless

The Variants

• Normal Wings vs. Vestigial Wings

http://www.schooltr.com/Teacher_Resources/Lesson_Plans/Lesson_Plan_images/Drowsy_Drosophila15.jpghttps://tbsgeneticsa.wikispaces.com/file/view/wild_vg.jpg/188678709/wild_vg.jpg

Wild Type Vestigial Winged

The Variants

• Normal Wings vs. White EyedWild Type White Eyed

http://www.brain.riken.jp/bsi-news/bsinews30/files/network0101-big.jpg

http://images.fineartamerica.com/images-medium-large/wild-and-white-eyed-fruit-flies-photo-researchers.jpg

Lab Setup• For today – we’ll call this Day 0 – you need to do

three things:– Decide on which cross you want to do and let me know

when I come to your table.– Set up your fruit fly tubes.• Get a plastic tube and a foam stopper.• Use a felt-tip pen to write your group’s names on the tube as

well as the genetic variant (wild type and vestigial wings/white eyes).– Don’t forget to decide which gender will be which.

• I will add a small layer of food (called white media) – you add an equal layer of water on top of it.

– Plug the tube and place it by the windows.

Background Drosophila Information

• Flies have been anesthetized by being refrigerated overnight, as have been the petri dishes in which they will be observed.– Being ectothermic, fruit flies’ metabolisms slow down to the

point that they cannot move.• Your goal in these initial observations is to get good at

sexing males and females, and at differentiating between wild type and mutant types.– Make note of how many individuals you observe with each trait

and what sex those individuals are.– Use the dissecting scope (stereoscope) – zoom is above the stage.– Make sure everyone in your group gets good at this.

How to Sex a Fruit Fly

• Differing Characteristics:• ♂– Dark, rounded abdomens

• ♀– Striped, pointed abdomens

• The “point” is an ovipositer – the egg-laying organ.

• Males also have a “sex comb” on their front legs, but it’s hard to spot.

http://ls.tcu.edu.tw/mdlin/Userdata/Image/male%20and%20female.jpg

http://www.stonybrook.edu/commcms/biology/images/Drosophila%20Male%20Female.jpg

Background Drosophila Information• The complete Drosophila life

cycle takes 10-12 days on average.– See lab sheet for more

information.– Disclaimer: I will refer to

adults as “hatching,” but in reality they metamorphose from pupae.

• Mating, as you might imagine, is hard to control.

• Furthermore, females store sperm and only release it when they lay eggs.

Drosophila Life Cycle

http://www.flinnsci.com/store/catalogPhotos/FB0361cat.jpg

Background Drosophila Information

• Since females can store sperm, we can’t control crosses if they’ve already had a chance to mate.– We need to ensure females have had no such opportunity

and that they can only mate with males we’re crossing with them.

• In order to control the experiment, then, we need to use virgins.– The adults we will be observing today as practice will be

killed afterward.– Tomorrow, we’ll separate the newly-hatched flies before

they reach maturity and mate with one another.

Lab Sequence

• Day 0:– We did this already – it was our initial set up of food

containers.• Day 1:– That’s today. We’re practicing sexing flies and identifying

phenotypes.• Work quickly while observing flies. You may keep the lid on your

petri dish if it makes you feel more comfortable.

– Adults removed, leaving their eggs to hatch and form our P generation.

• Seems like a good time to record this stuff in your notebooks.

Lab Sequence

• Day 2:– Set up our crosses by introducing the P generation parents to

one another.– You’ll need about 3-4 pairs from the newly-hatched

individuals.• Make sure you keep it consistent with gender – only one gender for

each trait.– As in, “wild type females with white-eyed males,” for example.

• These pairs will [hopefully] breed and give rise to the F1 generation.

– Of the ones I tap onto your petri dish, separate the individuals you need using a Q-tip.• I need the rest back ASAP. Get the lid on the petri dish in case they

wake up.

Lab Sequence

• Day 3:• Change the “3” according to whichever day it is.

– Kill the P generation adults. They’ve laid the F1 eggs.

• Day 4:– Observe the F1 generation and record your data.• How many individuals, how many male/female, how many male

variant, how many female variant, et cetera.

– Select 3-4 pairs of males and females.• You pick the traits to go with the sex.• It can be different from last time (if you want).

– They’ll give rise, ultimately, to the F2 generation.

Lab Sequence

• Day 5:• Change the “5” according to whichever day it is.

– Kill the F1 generation adults. They’ve laid the F2 eggs.

• Day 6:– Observe the F2 generation and record your data.• How many individuals, how many male/female, how many

male variant, how many female variant, et cetera.

• Day 7+:– Continue observing the F2 generation and increasing

the sample size.

Lab Sequence [General]

• Day 0: Set up tube.• Day 1: Practice sexing flies, kill the adults.• Day 2: Add newly-hatched pupae (young adults) to your own

tubes – they’re the P generation.• Day 3: P adults have laid eggs.– Kill P adults, wait for hatching.

• Day 4: Analyze the resulting (F1) phenotypes.

• Day 5: F1 adults have laid eggs.– Kill F1 adults, wait for hatching.

• Day 6: Analyze the resulting (F2) phenotypes.

• Day 7+: Continue observing/collecting F2 flies.

Intermission

Intermission

From F1 to F2

• On our first day of the transition between generations, we’re going to count all individuals from F1 – the ones currently in the tubes.– And kill them.– You’ll anesthetize them and then dump them into a petri dish

with a thin layer of ethanol.• Next class, we’ll select some newly-hatched individuals

and make them the F2 generation, placing them into fresh tubes with new food.

• For this first round of F1 data, record sex and phenotype of all individuals.

From F1 to F2

• When we move to the F2 generation, select a “handful” of flies and record sex/phenotype information.

• If you have the ability and choice, try to make males one phenotype and females another.– Either way, be sure to record this info.

Analysis and Conclusion Details

• Believe it or not, there’s room for a statistical test.• Following the analysis of the F1 generation, develop a

hypothesis for how the trait you’ve selected is inherited.– Is it autosomal dominant, autosomal recessive, sex-linked

(dominant or recessive)?

• For each mating generation (P and F1), perform a Punnett square and calculate the probabilities of each phenotype found in the offspring.– This is your null hypothesis.

Analysis and Conclusion Details

• So you’ll have expected amounts of offspring phenotypes from the Punnett Squares, but then you’ll also have actual recorded numbers of offspring phenotypes that formed.– What test does it sound like you’re going to use?– A chi-squared test! Yay you.

• Given the total actual amount of offspring that formed, compare observed (in the vial) phenotypes to expected (from the Punnett square) phenotypes.– Degrees of freedom? That depends.– Determine degrees of freedom from your Punnett square results.

• Remember, it’s possible outcomes minus one.

The Role of the Null Hypothesis

• Just for the record, in this case, you “want” to accept your null hypothesis.

• Not having a difference from your Punnett square means your hypothesis of inheritance mode is correct.– So accepting the null hypothesis, in this case,

means you also accept your own hypothesis, and vice versa.

Analysis and Conclusion Details

• For your lab notebook, put the chi-squared test (and the work) in the Analysis section.

• Put the discussion of the meaning of your chi-squared results in the conclusion.– Can you accept your null hypothesis?– Do the data not really line up with any conceivable

pattern of inheritance?– Could there actually have been a different kind of

inheritance at work?– Did any parent fruit flies accidentally stick around?– Could someone have mixed up male/female?

The Lab Report

• Put the whole thing together according to the informal lab report rubric.

• The report will be due one week following the conclusion of the experiment.