Lab 9: Gene Mapping, Crossing Over, Centromere Mapping (Sordaria

fimicola)

- Sordaria ascospore analysis (mapping gene distance from the centromere)

- Gene mapping in eukaryote (calculating distances between genes)

Reading Assignments:

Course materials on Blackboard.

Homework assignments:

Questions posted at the end of this worksheet.

To Do:

- Prepare slides of Sordaria perithecia and analyze the ascospores contained in the asci.
o Each person at a table should count 1 perithecium or 2 perithecia, and the table will collect
their data together

a. How many asci contain a parental order of ascospores?

b. How many asci contain ascospores representing crossing over?
c. How many total segregants were analyzed?

- The ratio of second division segregants to total asci = b / c

- The crossing over frequency = b / [c x 2]

- The distance from the spore color gene to the centromere is the frequency x 100

- Keep in mind that the crossover frequency is equal to half the frequency of second division
segregants, since only two of the four homologous chromosomes are involved in the crossover.

- Think of it this way: if 100% of asci were second division segregants, only 50% of chromosomes
would have been involved in crossing over. The second division segregant frequency therefore
overestimates crossing over by half.
Sordaria fimicola life cycle:

Sordaria is an ascomycete fungus that spends most of its life cycle in a haploid vegetative state.

However, under certain conditions, two haploid strains can be induced to undergo a sexual process in which
they fuse to form diploid zygotes.

Sordaria fimicola meiosis:

After a diploid zygote forms from two haploid strains, the cell undergoes Meiosis I, Meiosis II, and one round of
Mitosis (eight haploid cells). The cells are distributed linearly in an ascus (plural asci), and ~20 or so asci are
then assembled together in a perithecium.

Since the ascospores are haploid, their genotype = phenotype

The allele of the spore color gene can therefore be determined simply by looking at the color of each ascospore.

Non-crossing over: Crossing over:

Look carefully at the image above. Two haploid strains, each containing a different allele for spore color, have
fused to form a diploid zygote. Notice that if crossing over occurs between homologous chromosomes during
Meiosis I, the phenotypic sequence of spore color changes.

The left part of the figure demonstrates meiosis and mitosis of a zygote (2n). Without crossing over, four
ascospores on one side will be the same color. See the figure below (a).

The right pane illustrates a crossing over between the gene and the centromere. The recombination alters the
genotype sequence in an ascus, which will be observed as an altered color sequence. See the figure below, (b)
and (c).

Parental and Crossover Phenotypes:

(a) Non-crossing over (b) Crossing over (c) Crossing over

Linkage Analysis

Steps to performing linkage analysis:

1. Linkage or independent assortment?

 Linkage analysis is performed by examining phenotypic ratios of a test cross.

 If genes assort independently (non-linked): gamete production is random, and phenotypes are
present in equal amounts

 Linked: uneven distribution of phenotypes.

2. Which genes are linked?

 This applies when 3 or more genes are linked

 See the PowerPoint lecture for determining how many genes are linked

3. What is the gene order?

 Identify DCO (double cross over), whose phenotypes are rare

 Compare parental and DCO alleles.

 Among three genes, only the middle gene will be switched when DCO occurs.

4. Analyze genetic distance between adjacent genes one pair at a time

5. Determine the Coefficient of Coincidence and the Interference value

Practice problems – To be filled out in class

1. Construct a map of a chromosome containing 4 genes.

Some of the distances between these genes are listed below.

The distance between genes R and C is 10 units

The distance between genes C and P is 13 units
The distance between genes P and R is 3 units
The distance between genes S and C is 18 units
The distance between genes S and R is 8 units

What two genes would likely have the most recombination events between them?

What two genes would likely have the fewest recombination events between them?

2. In panthers, black coat (g) is recessive to golden coat (G), pink eyes (b) is recessive to blue eyes (B), and
short tail (l) is recessive to long tail (L). A female heterozygous for all three genes is test crossed to a
homozygous recessive male and the following offspring were obtained:

BGL 70
BgL 78
bGL 358
BGl 4
bgL 8
Bgl 344
bGl 70
bgl 68

a. How many genes are linked?

b. Which groups are the parentals?

c. What do the other groups represent?

d. What is the correct gene order?

 e. What are the distances between all of the linked genes?

f. What is the coefficient of coincidence?

g. What is the interference value? What does it mean?

3. An individual heterozygous for the following three alleles (XYZ) was testcrossed to a homozygous recessive
individual (xyz). The following offspring were generated:

XYZ 200
xyz 210
xYZ 215
Xyz 205
xYz 80
XyZ 90
xyZ 85
XYz 95

a. How many genes are linked?

b. Which allele groups are parental?

c. What do the other groups represent?

e. What are the distances between the linked genes?

 Genetics Lab: Homework assignment
Lab 9: Gene Mapping, Crossing Over, Centromere Mapping (Sordaria fimicola)

1. An individual heterozygous for three genes with the phenotype ABC is test crossed to a homozygous
individual (phenotype abc). The following offspring are observed:

ABC 100
abc 100
Abc 90
aBC 90
ABc 300
abC 300
AbC 350
aBc 350

a. How many genes are linked?

b. What genes are linked?

c. What is the distance between the two linked genes?

2. Sordaria is an ascomycete and is usually in a haploid state. Why are they used in the Genetics lab for
crossover analysis despite the fact that they are typically haploid?
3. Two strains of Sordaria are crossed with spore phenotypes a and b. The following table shows the number
of each type of ascus found. How far is the a/b locus from the centromere?

Ascus type [Number of Asci]

aabbbbaa [30]
bbaabbaa [74]
aaaabbbb [245]
aabbaabb [65]
bbaaaabb [43]
bbbbaaaa [323]

4. Two strains of Sordaria are crossed with spore color phenotypes + and s. The following table shows the
number of each type of ascus found. How far is s from the centromere?

Ascus type [Number of Asci]

++ss++ss [52]
ss++ss++ [65]
++ssss++ [120]
ss++++ss [130]
++++ssss [198]
ssss++++ [212]

5. During your analysis of Sordaria asci, you may have noticed some unusual patterns (3:1:1:3 and 5:3) which
cannot be explained by simple crossing over. These patterns are due to gene conversion. What is gene
conversion? Explain the process by which gene conversion can create these patterns.