BISC 219/2009: Mod 1 Lab 2

Lab 2: Scoring Autosomal vs. X-linked

Record in your lab notebook the number of WT, Dpy, Unc and Dpy Unc mutants by scoring the phenotype and removing that animal from the plate (flame the pick to remove the worm). If unlinked, you should see WT's, Dpy’s, Unc’s and Dpy Unc’s in a ratio of 9:3:3:1. If linked, you should see greater than 1/7th Dpy Unc’s among the mutant progeny.

You should now be able to conclude which strain is autosomal and linked, autosomal and unlinked and finally which strain has an x-linked gene and which one it is Dpy or Unc.

Mutant Hunt: Gene Mapping

In this exercise, you will progress through the normal sequence of events in forward (classical) genetics. You will first perform a mutant hunt, scanning a plate for rare mutants that occur among the background of wild-type animals. You will then pick your mutants to a separate plate, confirm their mutant phenotype, and begin genetic analyses that culminate with mapping the mutation. In truth, mutants will have been secreted on the plate by your devious instructor. Thus, while rare, they will be more frequently encountered than had you sifted through the second-generation progeny from mutagenized worms (as is the case with a real mutant hunt).

Once you have recovered your mutant and confirmed its phenotype (by examining its progeny) you will next perform two tasks in parallel. One of these is to backcross the mutation into wild type. This consists of crossing your mutant times wild type and recovering homozygous mutants in the F2 generation. Backcrossing is necessary to eliminate any secondary mutations that might modify the phenotypes you are studying. This is important because most mutant hunts employ heavily mutagenized P0’s. Backcrossing will also reveal X-lined mutations, as you recall from the previous exercise.

The second parallel task is linkage testing; you will determine which of the five autosomes (linkage groups) your mutation is located on. This is prerequisite to mapping. This is accomplished by determining the segregation behavior of your unmapped mutation relative to standard reference markers (e.g., mutations whose location is already known). Recall that unlinked mutations will segregate independently (your basic dihybrid inheritance as first observed by Gregor Mendel) whereas linked mutations will not.

In practice, linkage tests are performed using the following steps (where "m" represents your recessive mutant tested with reference marker "r"). The markers m and r must be distinguishable. Since homozygous mutant males usually won't mate, the desired trans double heterozygote is constructed by mating males heterozygous for one mutation (m/+) with hermaphrodites homozygous for the reference mutation (r/r). The genotypes of the F1 hybrids will be +m/r+ and ++/r+. We are only interested in the double heterozygote. The F1 hybrids containing only r are not useful. To select the m/r heterozygotes, we clone 4 or 5 individual F1's on small plates. We score the progeny of the F1 individuals (the F2) for linkage. Only F1 clones which segregate m/m homozygotes are scored. This should be 50% of the plates.

F2 progeny of each class are counted: m, r, mr double, and wild-type. If assortment is independent, progeny will be:

C. elegans General Information
Tools and Techniques
Lab 1: Welcome to C. elegans
Lab 3: Linkage Test and Backcross
Lab 4: Linkage Test, Backcross and Mapping
Lab 5: Mapping Part 2
Lab 6: Score!