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Three-Point Cross Aspergillus nidulans

 Group #: 50
 Names: Marleen van der Wiel & Mats Ewals
 Fill in the answers below each question

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Classical three-point cross with Asperigullas nidulans (ascomycetes)
After meiosis the meiotic products are contained in a sac (ascus) which makes it handy in genetics analysis.
The first genetic map was constructed by Aflfred sturtevant at age 21.

Parasex= recombination outisde the sexual cycle, it is mitotic recombination instead of meiotic
reconbination. You can also use this for mapping
Heterokaryosis= cell that contains multiple nuclei

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Abundant in the Netherlands which can kill you if your immune system is not functioning well.

Mutations are seen immediately

Can grow on a minimal medium (MM) because it is prototrophich and can produce everything from scratch
that it is needed. You can knock out genes that it is needed for the production to map the pathways and study
genes this way (called auxotrophic mutants)

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Experiment A
Unlinked and linked genes

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Genetic analysis, step by step
In the cross to be analysed here, the parents carry different alleles for four genes, the isolates are otherwise identical
(i.e. isogenic). In other words, four contrasts can be analysed.

1. yA+, biA1, pabA+, acrA1 X 2. yA2, biA+, pabA1, acrA+

1. A monogenic contrast (First Mendelian Law)

First, we will analyse one contrast from the cross between the parents: the spore colour (green, yA+, vs yellow, yA2).
Simplified genotypic notation of the cross: yA+ x yA2.

Questions
1) What has been the genotype of the ascus mother cell of this cross (=diploid meiocyte, see Fig 1. in introduction
page A2)? Ya+Ya2
Ya+, bia1, pabA+, acrA1//ya2, bia+, aba1, acrA+
2) Draw schematically a meiocyte with two homologous chromosomes with their chromatids on which the colour
gene is located at metaphase of meiosis I (M-I). Indicate the two different alleles in your drawing as well as the
centromeres.

Two homologous chromosomes, crossing over between non sisted chromatids, four chromatids and the centromere

3) Use the χ2 test to test whether the inheritance of yellow and green conidiospores is likely due to a single
Mendelian gene.

Phenotype Genotype Observed Expected (O-E)2 ( O−E )2

number (O) number (E) E
green yA 39 40 1 0.025
yellow yA2 41 40 1 0.025
( O−E )2 = 0.05
Total Chi−square value : (0.025+0.025)
E
a) How many progeny are green, how many yellow? Fill in the observed numbers.
b) Define the null hypothesis of equal segregation of the two alleles into the haploid progeny (first law of
Mendel).
Equal segregtion of the 2 alleles yA+ and yA2(green: yellow progeny expected= 1:1)
c) Calculate the expected number of yA+ and yA2 progeny assuming the null hypothesis (fill in the table).
d) Calculate the χ2 value from the formula χ2= ∑(obs-exp)2/exp. Note that the bigger the difference gets between
the observed and expected data, the higher will be the χ 2 value. With one degree of freedom (# of classes - 1),
the chance of obtaining a χ2 value of 3.84 or higher is <0.05. In the case of a χ 2 value > 3.84 the null
hypothesis is rejected (see χ2 value table, e.g. Griffiths et al. 2015 p97), with lower value the null hypothesis is
not rejected (but not proven).
e) Draw your conclusion. Our H0 is not rejected, so the alleles for the gene colour our expected to segregate
in a 1:1 ratio

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f) Now analyse the other contrasts in the same way and conclude which are likely monogenic Mendelian
contrasts.
g)
Gene Progeny number with Consistent with monogenic Mendelian segregation?
Wild-type allele Mutant allele (Indicate χ2 value)
pabA 39 41 pabA+ = 0,025 pabA2 = 0,025 YES
biA 42 38 biA+ = 0,1 biA2 = 0,1 YES
acrA 34 46 acrA+ = 0,9 acrA1 = 0,78 YES

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X

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2. Two contrasts, no linkage (Second Mendelian Law)

If two loci are located on different chromosomes they segregate independently according to the second law of Mendel
(“independent assortment of genes”), their recombinant frequency will be 50%, and therefore genetically unlinked.
As an example, we will analyse the unlinked colour and fungicide resistance genes:
Simplified genotypic notation of the cross: yA+, acrA1 X yA2, acrA+

Questions
1) Score the phenotypes of the 80 progeny, each colony separately, in the following phenotype table: A mutant
phenotype will be indicated with an X in the table, thus a yellow colony will get an X in the first column, an
acriflavin-resistant colony will get an X in the second column. The 20 colonies of Plate 1 are already filled in as
an example.

Table of Phenotypes
Plate 1 Plate 2 Plate 3 Plate 4
Colony yA2 acrA1 yA2 acrA1 yA2 acrA1 yA2 acrA1
1 X X X X
2 X X
3 X X X X X
4 X X X X X
5 X X X X
6 X X X X X
7 X X X
8 X X X X
9 X X X X X X X
10 X X X X X
11 X X
12 X X X X X X
13 X X X X X
14 X X X X X X X
15 X X X X
16 X X X X
17 X X X X
18 X X X X X
19 X X X
20 X X X

2) Fill in the Table below. Indicate which four genotypes and at what number are among the progeny in the
phenotype table? Also, indicate which genotypes are parental and which are recombinant.
Genotype Number Parental or Recombinant?
yA acrA1
+
22 P
yA+acrA+ 17 R
yA2acrA+ 17 P
yA2acrA1 24 R

3) Are the numbers in each genotype class approximately the same, as expected? YES~1:1:1:1
4) Calculate the recombinant frequency. ((17+24)/80) x 100 = 51,25%
5) Does the recombinant frequency comply with the expectation of the second law of Mendel? (If in doubt you can
do a χ2-test). YES

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3. Two contrasts, linkage

Genes on the same chromosome will reveal linkage if they are not too far from each other. Thus, they will not follow
the second law of Mendel! Let us look again at our cross:

yA+, biA1, pabA+, acrA1 X yA2, biA+, pabA1, acrA+

1) We will first analyse colour and pab-auxotrophy. A visual inspection of the photographs may give already an idea
about these two genes: What is the colour of the majority of the colonies on the SM-pab (genotype pabA+) test
plates? Green Which colour is most abundant among the pabA1 colonies? Yellow
2) Are the two genotypes that appear to be present at the highest numbers recombinants or parental genotypes?
Parental (yellow paba-def; green not def for pab suggesting linkage
3) Analyse in the same way colour and biotin auxotrophy. Most biotin deficient porgeny (biA1) is green, biA+
mostly yellow
4) Next, judge the contrasts biA1/biA+ and pabA1/pabA+. Complete the following Table of Phenotypes:

Plate 1 Plate 2 Plate 3 Plate 4

colony pabA1 biA1 pabA1 biA1 pabA1 biA1 pabA1 biA1
1 X X X
2 X X X X
3 X X X X
4 X X X
5 X X
6 X X X
7 X X X X
8 X X X X
9 X X
10 X X X X
11 X X X X X X
12 X X X X
13 X X X X
14 X X
15 X X X X X
16 X X X X X
17 X X X X X X X
18 X X X
19 X X X X
20 X X X X X

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5) What are the expected genotypes for the markers biA and pabA? How many colonies do you find of each? Fill in
the Table of Genotypes.

Genotype Parental or Number observed

Recombinant?
pabA1biA+ P 29
pabA1biA1 R 11
pabA+biA+ R 13
pabA+biA1 P 27

The recombinant frequency is used as an index of the genetic distance between markers.
The mapping unit (1 m.u. or 1 centiMorgan) is defined as a recombinant frequency of 1 percent.

6) Apply the 2 test for testing linkage of the two markers.

a) H0=no linkage (independent assortment of the two genes). We could test this H 0 with expected values of 20 for
each of the four genotypes, but alleles may have different viability and therefore we will here adjust for
differences in allele frequency among progeny.
b) Fill in the observed values for the four genotypes and calculate the total numbers of the alleles.

Observed pabA+ pabA1 Totals

biA+ 13 29 #biA+

42

biA1 27 11 #biA1

38

Totals #pabA+ #pabA1 # progeny, N

40 40 80

Next make a contingency table in which you fill in the expected numbers based on the allele frequencies among the
progeny. E.g. the expected frequency of biA+pabA+ =(#biA+* pabA+)/N etc.

Expected pabA+ pabA1 Totals

biA+ 21 42*39/80= 21 42*41/80= 21,5 #biA+

20,5
42

biA1 19 38*39/80= 19 38*40/80= 19 #biA1

18.5
38

Totals #pabA+ pabA1 # progeny, N

40 40 80

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 Now you can calculate the 2 value, use df is 1 (=(number of rows-1) * (number of columns -1)) and draw your
conclusion.

((13-21)2/21) = 3.0476
((29-21)2/21) = 3.0476
((27-19)2/19) = 3.3684
((11-19)2/19) = 3.3684
Total = 12.832
High Chi-squae value 12.832  3.8; independent assortment of these genes is highly unlikely
We can reject the H0 hypotheses and it can be expected there is linkage between these alleles.
7) If these genes are on the same chromosome, what is the percentage of recombinants, and thus the distance in cM?
30%, that also means 30 cM. (13+11/80*100= 30)

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4. Three linked loci (three-point cross)

Thus far we found three loci that are linked: yA, pabA and biA. For these genes, you will construct a linkage map.

1. You have to know the complete genotype of each colony. Check the following Table of Phenotypes.

Plate 1 Plate 2 Plate 3 Plate 4

yA pabA biA yA pabA biA yA pabA biA yA pabA biA
2 1 1 2 1 1 2 1 1 2 1 1
1 X X X X
2 X X X X X
3 X X X X X X
4 X X X X X X
5 X X X X
6 X X X X X X X
7 X X X X X
8 X X X X X X X
9 X X X X X X X
10 X X X X X
11 X X X X X X X
12 X X X X X X X X
13 X X X X X
14 X X X X X X
15 X X X X X
16 X X X X X X X X
17 X X X X X X X
18 X X X X X X
19 X X X X X X X
20 X X X X X

2. Fill out the Table of Genotypes

Recombinant for pair of markers P, sco or

Genotype Number yA-pabA pabA-biA yA-biA dco
yA2 pabA1 biA1 2 X X Sco
region 1
+ + + 3 X X Sco
region 1
yA2 + + 6 X X Sco
region 2
+ pabA1 biA1 6 X X Sco
region 2
yA2 + biA1 1 X X Dco
(1+2)
+ pabA1 + 1 X X Dco
(1+2)
yA2 pabA1 + 32 P (no co)
+ + biA1 29 P (no co)
number 80 14 17 7
recombinant frequency 14/80*100- 17.80*100= 7/80*100
17,5% 21.25% %= 8.75%

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3. What frequencies for the 8 genotypes would you expect to find if all three markers were unlinked?
1/8 for all genotypes
4. Mark, with an X in the Table, for each genotype whether there has been recombination in a given region.
5. Indicate which genotypes are the parentals (P), single crossovers (sco) or double crossovers (dco).
6. The different genotypes have different frequencies. What do you conclude from this? The frequencies can be
explained by the fact that there are single cross and double cross overs. The fact that double cross overs have a
lower chance of happening than single cross overs might explain the fact that there are different frequencies.
Linkage of the three genes (parental genotypes more frequent because genes tend to stay together due to linkage)
7. Among the genotypes, you will find pairs of ‘mirror types’ (reciprocals). The frequencies of such reciprocal
genotypes are expected to be similar. Explain why. When a cross over happens, there will arise two new
recombinants, which are called mirror types. This frequency of mirror types will be the same due to the fact
that certain parts of the chromosomes are mixed at the same time.
They result from the same meiotic event.
8. The double-crossover types can be used to find the order of the genes:
Suppose the genotype of the diploid meiocyte is
a b c

a+ b+ c+
then the double-crossover types will be a b+ c and a+ b c+, thus the gene in the middle took its allele from ‘the other’
parental chromosome.
What has been the genotype of the meiocyte in the Aspergillus cross? Compare the genotypes of the double-crossover
types with the parental genotypes and make your conclusion about the order of the genes.
yA2, pabA1, biA+// yA+ pabA+ biA1, yA2 changes poistion by the dco (when comparing parents and deco’s) and is
in the middle
9. Calculate the percentage recombinants in each area.
10.Draw the genetic map with the order and distances between each of the three loci. (Note that you have no
information on the position of the centromere!)
pabA-17,5- yA-8.75m.u.- biaA
11.Why is the distance, found between the two outside markers, not equal to the sum of the two smaller regions? Dco
can involve the same chromatids so that for the outside markers there is no recombination seen even though
there has been 2 co’s. When looking at the 2 smaller regions we do see the double crossovers and countthem
for each region but these are unseen considering pab/bi.
What is the best estimation of the genetic distance? The best estimation of distance includes also the ‘unseen’
co’s. so 17.5+ 8.75 is the better estimation of the distance between pabA and biA.

The genetic map length of a chromosome in cM (or map units) reflects the mean number of meiotic crossover
events for that chromosome: Each crossover contributes 50cM, so if on average 2.3 crossovers occur per meiosis, the
map length is 2.3*50cM=115cM.
For markers close together on a chromosome crossover frequency will be low and all recombinants will be the result
of a single crossover. If e.g. 2% recombinants are found for adjacent markers, the genetic distance is 2 cM. However,
for markers showing a higher recombinant frequency, the recombinant fraction does not properly reflect crossover
frequency due to double crossovers that may also occur, but do not increase recombinant fraction. Therefore, if you
find 30% recombinants for a region, the actual distance is >>30cM.
In order to correct for unseen crossovers Haldane developed a mapping function to more precisely calculate m, the
mean number of meiotic crossovers in that region. RF=1/2 (1-e-m) → m= -ln(1-2RF). From m, the corrected distance
in cM can be calculated: m*50cM.
So, 30% recombinants -> RF=0.3 -> m=0.91 -> 45.8cM

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12. Parental (non-recombinant) progeny may result from absence of crossover, but also from double crossover (see
Figure). Calculate the corrected recombinant map units for the three markers.

recombinant
Observed

fraction

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