Lab 7 Heredity. Is there a fly in here?

Transcription

1 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).

2 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.

3 Phenotypes Wild Type Curly Winged White Eyed Vestigial Winged Yellow Ebony Antennapedia (head-legs) Eyeless Orange Eyed

4 The Variants Wild Type Normal Wings vs. Vestigial Wings Vestigial Winged

5 The Variants Wild Type Normal Eyes vs. White Eyed White Eyed

6 Lab Setup For today we ll call this Day 0 you need to do this: 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 (masking tape available) 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 a roughly same-sized layer of water on top until it dampens but isn t runny. Include, if possible, a piece of plastic mesh. Plug the tube and place it by the windows.

7 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. Use the dissecting scope (stereoscope) zoom is above the stage. Make sure everyone in your group gets good at this.

8 How to Sex a Fruit Fly Differing Characteristics: Dark, rounded abdomens Striped, pointed abdomens The point is an ovipositor the egg-laying organ. Males also have a sex comb on their front legs, but it s hard to spot.

9 How to Sex a Fruit Fly Female Male

10 Background Drosophila Information The complete Drosophila life cycle takes 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.

11 Drosophila Life Cycle

12 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. Or saved. We ll see. But they always need to be removed before next class, when we ll separate the newly-hatched flies before they reach maturity and mate with one another.

13 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.

14 Lab Sequence Day 2: Set up our crosses by introducing the P generation parents to one another. You ll need about 3-4 males and 3-4 females from the newlyhatched 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 F 1 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.

15 Lab Sequence Day 3: Change the 3 according to whichever day it is. Kill the P generation adults. They ve laid the F 1 eggs. Day 4: Observe the F 1 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 like last time. 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 F 2 generation.

16 Lab Sequence Day 5: Change the 5 according to whichever day it is. Kill the F 1 generation adults. They ve laid the F 2 eggs. Day 6: Observe the F 2 generation and record your data. Day 7+: How many individuals, how many male/female, how many male variant, how many female variant, et cetera. Continue observing the F 2 generation and increasing the sample size.

17 Lab Sequence [General] Day 0: Set up tube. Day 1: Practice sexing flies, kill the stock adults. Day 2: Add new immature stock adults to your own tubes they re the P generation. Day 3: By now, P adults have laid eggs. Kill P adults, wait for hatching. Set up new tube. Day 4: Analyze the resulting (F 1 ) phenotypes. Cross. Day 5: By now, F 1 adults have laid eggs. Kill F 1 adults, wait for hatching. Day 6: Analyze the resulting (F 2 ) phenotypes. Day 7+: Continue observing/collecting F 2 flies. 1 Week Intermission 1 Week Intermission

18 From F 1 to F 2 On our first day of the transition between generations, we re going to count all individuals from F 1 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 F 2 generation, placing them into fresh tubes with new food. For this first round of F 1 data, record sex and phenotype of all individuals.

19 From F 1 to F 2 When we move to the F 2 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.

20 Analysis and Conclusion Details Believe it or not, there s room for a statistical test. Following the analysis of the F 1 generation, develop a hypothesis for how the trait you ve selected is inherited. Is it autosomal dominant, autosomal recessive, sexlinked (dominant or recessive)? For each mating generation (P and F 1 ), perform a Punnett square and calculate the probabilities of each phenotype found in the offspring. This is your null hypothesis.

21 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.

22 About Degrees of Freedom Degrees of freedom? That depends. If you think it s autosomal: There are two different phenotypes (wild and vestigial/white), so there are two outcomes (minus one). If you think it s sex-linked: There are two different sexes and two different phenotypes (wild and vestigial/white), so there are four outcomes (minus one).

23 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.

24 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 chisquared 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?

25 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.