C. Tetrad analysis in Sordaria fimicola.

Group #: 50
Names: Marleen van der Wiel & Mats Ewals

In some fungi single meiosis can be studied really well. In ascomycetes the ascus contians the
meiotic products and remain in the same sack. By studying this sack we can study what
meiosis produced, this can be in an ordered and unorderer fashion. In the ordered fashion you
are able to see what exaclty happened during meiosis.

This can be seen in:

- Sordaria fimicola
- Neurospora crassa
- Podospora anserina

Mono-tetrad analysis: single gene segregation

You can also show Mendels first law in this way.

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Ordered tetrads analysis in Sodaria fimicola
When the tetrads are ordered you have more information then a single segregation. They are
formed that the spindeles are ormed in the same direction and there is very predictable spore
formation. In this fungus you have a post meiotic division. This can be used for centromere
mapping

without cross over

with cross over

- Centromere of homologs segregate at MI (FDS, or first division segregation)
 Centromere is a reference point (marker
- Markerslinked to the centromere also show MI (FDS)
 4A:4a pattern
- Sister chromatids segregate at MII
- Corssove between centrome and a marker results in MII or second division
segregation (DSD) of that markers
- MII asci show 2A:2a:2A:2a pattern
- Each MII ascus contains 50% recombinants spores
- Distance between the marker and the centromere  fraction MII*50cM

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- If A/a and B/b are linked pd>NPD; if unlinked PD~NPD
- Based on the 2 markers (using fractions of NPD and T)
o Recombinant freuqency, RF= 1(1/2T+NPD)= 50(T+2NPD)

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Fill in the answers below each question

A. Monohybrid tetrad-analysis: Mapping a gene relative to the centromere

1. Score ~100 asci-arrangements and fill in the first column of the Table (you may cooperate with
other groups if needed).

Segregatio Gene conversion

n
Tetrad pattern # asci MI or MII with mismatch repair without mismatch repair
●●●●○○○○/○○○○●●●● 4:4 51 MI
●●○○●●○○/○○●●○○●● 2:2:2: 27 MII
2
●●○○○○●●/○○●●●●○○ 2:2:2: 24 MII
2
○○○○○●●●/●●●●●○○○ 5:3 1 X (asymmetrical)
○○○○●○●●/●●●●○●○○
●●●○●○○○/○○○●○●●● 4:4 3 X (symmetrical)
○○○○○○●●/●●●●●●○○ 6:2 2 X (asymmetrical)

2. Study the figures C1a and b, about when during meiosis the alleles are segregating, i.e. at MI or at
MII. Next, indicate in the Table (last column) for the first three tetrad patterns whether the hyaline alleles
segregate at MI, or at MII. We will deal with the last three patterns later.
3. a. When do the centromeres of the homologous chromosomes segregate? Always MI, always MII
or sometimes MI sometimes MII? It happens during the MI but not in the MII, here the chromatids
segregate, so always MI.
b. Explain why this known segregation of the centromeres allows you to identify which asci resulted
from a meiocyte with a crossover between the centromere and the marker. We know that the
centromere segregates in a M1 pattern and if this differs from the allele segregation (alleles show
M2 segregation) than we can conclude that crossover happened.

Since centromere of the homologous chromosomes segregate at MI, the identical alleles on the sister
chromaitds are connected to the same centromere and will co-segregate at MI to the same pole (4:4
pattern). Therefore, a high fraction MI indicates tight linkage of that marker to the centromere. IF
there is a cross over at MI, identical alleles will move to opposite poles and only at MII the different
alleles segregate (2:2:2:2 pattern)
c. What fraction of spores in an ascus resulting from crossing over is recombinant? 50%, in a crossover
the octad shows a 2:2:2:2 segregation with 50% recombination
4. Thus, the fraction of MII asci * 50cM reflects the distance of a marker to its centromere. Now
calculate the distance of the hyaline marker to the centromere, neglect the last three abnormal asci.
25,5%cM (0.5*51= 25,5cM)
5. The figure shows the fraction MII segregation for markers on a chromosome arm of Sordaria. The
fraction asci with MII for markers plateaus at a maximum of ~2/3, even for markers far from the
centromere, i.e. are unlinked to it and undergo multiple centromere proximal crossovers. Thus, only for
markers relatively close to the centromere a reliable map distance to the centromere can be determined.
How can this be explained?

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Because there is a chance there happens more than one centromere proximal crossovers between
the marker and the centromere. When this happens, you can’t make a reliable map.
When crossing over is rare, MII will properly reflect the fraction meiocytes with a single crossover.
The fraction*50cM is then a good estimation of the distance in cM. At larger distances when most
meiocytes have one or more crossovers the distribution of the four alleles in the tetrad becomes
random with respect to the centromere. The statistical outcome of random filling of the ascus with
the four alleles in a row (2A and 2a) is an expected 2/3 asci with MII and 1/3 with MI pattern. This
is because when a chromatid with A is directed to one pole, there is a 2/3 chance that a chromatid
with the a allele (2 of the remaining 3 allees are a) will also move to that pole (MII), but only a 1/3
chance that the A allele follows (MI).

6. Now consider the last three ascus types with abnormal segregation pattern. Watch the movie on
gene conversion and the Meselson-Rading model of recombination (and Figure 3 in the review on
meiotic recombination Baudat e.a. NRG 2013) and fill in the Table which of these result from gene
conversion with/without mismatch repair.
B. Dihybrid tetrad-analysis: Mapping two genes relative to the centromere
7. In tetrad analysis with two (or multiple) markers we can ask the following questions:
(i) How far are the markers from their centromere?
(ii) How can be concluded that markers are on the same or a different chromosome?
(iii) And if on the same chromosome, what is their order relative to the centromere?

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Let us consider a dihybrid cross between two Sordaria strains AB and ab that resulted in the following
1000 tetrads:
Ascus type (PD = parental ditype, carrying 2 parental genotypes; NPD = non-parental ditype, carrying
2 recombinant genotypes; T = tetratype, carrying all 4 genotypes)
Spore pair PD / NPD / T
PD NPD T T NPD PD T
1+2 AB Ab AB AB Ab AB Ab
3+4 AB Ab Ab aB aB ab aB
5+6 ab aB aB Ab aB AB AB
7+8 ab aB ab ab Ab ab ab
Number 804 1 92 5 1 92 5
MI or MII for Marker A? MI MI MI MII MII MII MII
MI or MII for Marker B? MI MI MII MI MII MII MII
(i) How far are the markers from their centromere?
a. Calculate the distance of A to its centromere from the fraction MII of marker A.
5+1+92+5 = 103, 103 x 0,5 = 51,5 cM,
first calculate the fraction so 103= 10.3= 0.103*0.5= 5.15cM
b. Likewise, calculate the distance of B to its centromere. 92+1+92+5 = 190, 190 x 0,5 = 95 cM
= 0.190*0.5= 9.5m.u.
c. Draw the three possible alternative maps based on the distances of A and B to their centromere (i.e. on
different chromosomes or on the same/different chromosome arm).

(ii) How can be concluded that markers are on the same or a different chromosome?
d. To decide whether A and B are on the same chromosome we can now look at the frequency of the
ascus types. Fill in the Table above for each ascus what type it is: PD, NPD or T.
e. Compare the PD and NPD fraction and conclude whether the genes are linked to the same centromere
or not. Explain your answer.
They are linked to the same centromere. This is because you would expect more recombinants if
they were unlinked.

Asci 1 and 6 show PD: 898 in total

Asci 2 and 7 show NPD: 2 in total
Fraction PD>NPD
If unlinked, PD would equal NPD (independent re-assortment of chromosomes);

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If linked, NPD for linked genes results from dco including all 4 chromatids; sinds NPD<PD we can
conclude linkage.

(iii) And if on the same chromosome, what is their order relative to the centromere?
For this, we need to determine the recombinant frequency for A and B.
f. Indicate the recombinant progeny in the Table (e.g. by highlighting).
g. Note that T asci carry 50% recombinants and NPD asci 100%. From this calculate the recombinant
frequency (in %) for A/a and B/b by just counting or use the formula derived on this principal:
RF=50*(T+2NPD) cM. Where T and NPD are the fractions of these types.
RF A/a & B/b = 0,5 x (102 + 4) = 53%.
RF= 0.5(102+4)/1000=5.3%= 5.3 m.u.
h. Again, when markers are not very tightly linked, double crossovers may have occurred. Taking such
unseen crossovers into account, Perkins1 derived the following formula:
corrected map distance = 50*(T+6NPD) cM.
Calculate the corrected distance between A and B using this formula from the fraction T and NPD asci.
RF A/a & B/b = 0,5 x (102 + 12) = 57%
RF= 50*(102=6*2)/1000= 5.7%= 5.7 m.u.
i. Is this corrected distance higher or lower than the uncorrected distance? Explain. It is higher, because
unseen cross-overs are now calculated into the frequency
j. Draw the genetic map.

k. Make a drawing (with all four chromatids and the two markers and their centromere) to explain the
origin of the second ascus from the left in the Table.

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l. You may also have wondered why some of the T asci are rare and others more common. Can you
explain this?
You see more often two capital A’s together in comparison with capital B’s because marker B is
further away from the centromere, so you would expect more cross overs between centromere and
B instead of the centromere and A.

Write a provisional report answering the questions. Upload on Brightspace today

before you go home. After the feedback lecture please upload your finalised report.
Challenging question: Explain why the corrected formula has 6NPD i.s.o. 2NPD?
1
Perkins 1949 Genetics 34: 607

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