Goals of the lab:

• Determine whether the mutant phenotype we observe is dominant or recessive to the wild type phenotype.
• Determine whether goi-1 is sex-linked or autosomal by analyzing progeny.
• Design and set up crosses to determine the chromosomal linkage group of goi-1.

Analyzing the phenotypes of progeny

In the last lab, we crossed our mutant hermaphrodites to wild type males. By observing the phenotypes of their progeny, we can learn whether our mutant phenotype is dominant or recessive to the wild type phenotype, and whether the gene causing the phenotype is located on an autosome or the X chromosome.

TO DO:

• Retrieve your crosses from the incubator and observe the progeny. Discuss the following with your lab partner: What is the genotype of these worms? How many copies of goi-1(219) do they carry? How many copies of goi-1(+)? What would you expect to see if the mutant allele were dominant? What if it were recessive? Record your observations and conclusions in your lab notebook.
• Take a picture of the progeny to support your conclusion (you will use this image in a figure in your homework).
• Next consider what you would expect to see if goi-1 were on an autosome versus the X-chromosome and discuss with your lab partner. Remember that males have only one X chromosome and are therefore hemizygous for any X-linked genes. (These predictions should be in your cross design from Lab 2). Record your observations and conclusions in your lab notebook.
• The phenotype of the male progeny are the most compelling evidence to indicate whether our gene of interest is on an autosome or the X. Take a picture of the male progeny to support your conclusion (you will use this image in a figure in your homework). If your first image is of a male, there is no need to take a second one.
• DO NOT discard these worms! We will use them in our next step.

Autosomal linkage analysis using reference markers

If our gene of interest is not located on the X chromosome, we next need to determine which chromosome it is on.

We can do this by determining whether our gene segregates independently from known reference markers, or mutations whose locations are already known. For example, if goi-1 segregates independently from unc-104, a reference marker on Chromosome II, we can determine that it is unlinked to unc-13 and therefore NOT on Chromosome II. In contrast, if it fails to segregate independently from unc-32 on Chromosome III, goi-1 is likely linked to unc-32 and therefore also on Chromosome III.

When writing the genotype of two different genes, genes in different linkage groups (chromosomes) are separated by a semicolon (;), while genes in the same linkage group are not. Chromosome numbers are written in Roman numerals after each gene or genes in the same linkage group.

For example, unc-104(e2184)/unc-104(e2184) II ; dpy-17(e164)/dpy-17(+) III refers to an animal that is homozygous for unc-104(e2184) on Chromosome II, and heterozygous for dpy-17 on Chromosome III with one mutant e164 allele and one wild type allele.

unc-32(e189) dpy-17(e164)/unc-32(+) dpy-17(+) refers to an animal that is heterozygous for dpy-17 as above as well as heterozygous for unc-32, carrying one e189 allele and one wild type allele. However, since both unc-32 and dpy-17 are located on Chromosome III, they are written together without the separating semicolon. This notation further indicates that both of the mutant alleles are present together on one homolog (on the left of the slash) while both wild type alleles are present together on the other (on the right of the slash). We refer to this as being in cis, or on the same homolog. The alternative is to have two alleles in trans, or on opposite homologs. Such a genotype would be written as unc-32(e169) dpy-17(+)/unc-32(+) dpy-17(e164). This difference is important, since predictions about offspring genotypes and phenotypes will vary based on the parental and recombinant gametes.

Designing an experimental cross

When using reference markers, it is crucial that the phenotype of the reference strain is visibly distinguishable from that of the unknown mutation. In this case, we will use Uncoordinated (Unc) mutants, which you observed in Lab 1. These reference strains are maintained as homozygous hermaphrodites through self-fertilization; there are no males on the plates. Due to their impaired movement, Unc males are unable to mate; therefore, males homozygous for the reference marker alleles cannot be used to set up crosses.

We have the following reference strains available. The Roman numeral in parentheses indicates the chromsome on which the gene is located.

• unc-13(e51) (I)
• unc-104(e1265) (II)
• unc-32(e189) (III)
• unc-17(e245) (IV)
• unc-76(e911) (V)

You will also have our unknown mutant hermaphrodites (homozygous for the mutant allele), and N2 (wild-type) males and hermaphrodites.

TO DO:

• With your partner, design a cross using the available worms listed above that will enable you to determine which reference marker is linked to our gene of interest.
  • NOTE: This will require more than one generation of worms! Remember that homozygous mutant males are unable to mate. In your design, indicate the genotypes of the worms you will mate, the genotypes of the gametes they can produce, and the genotypes and phenotypes of the resulting progeny. What ratio of phenotypes would you expect to see as a result of independent assortment? How is this different from what occurs when genes are linked? You should show one example cross in which you observe linkage and one example in which you observe independent assortment.
• Have your instructor check your strategy before moving on.
• Obtain five mating plates and two transfer plates per pair of lab partners.
• For each mating, transfer 3-4 heterozygous males (goi-1(219)/goi-1(+); unc*(+)/unc*(+) from the cross you initiated in Lab 1 onto a transfer plate to allow them to dissociate from any eggs or young larvae, then transfer them to a mating plate.
• Next transfer three or four L4 hermaphrodites that are homozygous for one of the five reference markers.
• Label your crosses with your PURPLE sharpie, including today's date and the description of the cross you just set up (be careful not to mix up your crosses!).
• Repeat for each of the reference strains. Place your crosses in your group's box and store it in the incubator at 23°C.

To Do 3-4 Days After Lab:

Linkage analysis: producing the F2 generation

The F1 progeny produced from the crosses you set up in Lab 2 should contain some animals that are heterozygous for both the reference marker and our gene of interest. However, because the father was heterozygous for our gene of interest, some F1 progeny will have inherited his wild-type allele, and will carry only a mutation in the reference marker gene. Since we cannot distinguish between these animals by their phenotype, we will have to self-fertilize multiple hermaphrodites from each strain.

TO DO:

• Working with your partner, obtain 20 maintenance plates from your lab days bin at the back of the room. Maintenance plates differ from mating plates in that the spot of bacterial food is bigger, providing an adequate food supply for more worms. Since the F1 hermaphrodites will self-fertilize, there is no need for them to find each other on the plate.
• From each cross, transfer four L4 hermaphrodites to separate maintenance plates, one worm per plate. It is EXTREMELY IMPORTANT that these hermaphrodites are L4 and not adult. Older worms may have already mated with the males on the plates, which would confound your F2 results analysis. Have your partner double check your worm identification!!
• Label your self-crosses with your PURPLE sharpie, including the expected genotype of the worm and the date. Place your self-crosses in your group's box, and store it in the incubator at 23°C.

With your partner, answer the following questions:

• To determine in what chromosome or linkage group our mutation is located, you first had to rule out the x chromosome. How did you do that?
• How many chromosomes can our mutation be on?
• How many of our crosses with unc reference strains will show independent assortment?
• How many of them will show linkage?
• What will independent segregation score like in these progeny? Why?