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Linkage analysis and QTL mapping using Recombinant Inbred

Line population of Arabidopsis thaliana
Group #: 50
Names: Marleen van der Wiel & Mats Ewals

Fill in the answers below each question

QTL (quantitative trait loci)
Phenotype with wide distribution range (polygenic), traits that can be affected by many genes.
Example: size
You see these kinds in all kinds of organisms, and you can link these traits to certain genomic
positions that may explain them. So we try to find what area of the genome correlates with a
particular trait.

Arabidopsis thaliana:
Main plant model organism
Reasons:
- Global distribution; adapted to all kinds of environmental variables, useful to study
trait/genes involved in those traits. Can be applied to other species
- Small, simple genetics, many seeds, whole genome available
- Phenotypic variation

Population development: RIL

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 Material & Method
The following materials are available on Brightspace:
- A RIL collection of 74 individuals (some plants could not be fully scored) was screened for
segregating molecular markers. Pictures of the gels are available for:
- CAPS marker (GAPB)
- Five AFLP fingerprints (AFLP464C, AFLP269C, AFLP204C, AFLP162C and AFLP116C).
- An excel scoring list for the six markers in which flowering time is already indicated on Blackboard
(Exp H Analysis form Students.xlsx). Work through the questions below to guide you to fill in the
Excel form and analyse the data.
- Note: the molecular variants (A or B) are given for only the first plant. It means that presence or
absence of a DNA band can be A (or B). Don’t use the traditional notation of A and a for dominant
and recessive alleles, as this is error prone (e.g. autocorrect functions in Word or Excel).

Conclusions.
Answer the following questions.

1. The figure below indicates the formation of Recombinant Inbred Lines (RILs) from a cross
between two homozygous Arabidopsis plants P1 and P2 (2n=10). The resulting F1 is then
selfed 7 times. Each bar represents a chromosome.

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 Both the paternal and maternal gamete undergoes meiosis, however this is not likely to be
at exactly the same place so in the first few generations you see that each chromosome is
different. In generation 7 most of the genome is homozygous due to selfing
a. Colour the chromosomes of the F1 to indicate its genotype
b. Indicate what two different RIL lines could look like after 1, 2 and 7 selfings by colouring the
pattern of the chromosomes in the figure.
c. Study in Fig H1 (top left part) the cartoon indicating selfing of the F1 leading to a heterogeneous
inbred family (HIF). Given that male meiosis shows on average 2 crossovers per chromosome and
female meiosis 1 co/chromosome, what is unrealistic about the F2 chromosomes drawn here?
In a F2 generation, they display too much cross-overs drawn
The second chromosome shows mirror types, this can happen but the chances are unrealistic.
d. Also explain why the 3rd RIL derived from the F2 population in Figure H1 is also not very
realistic.
The 3rd RIL displays too much cross-overs
After selfing it went from homozygous red to homozygous blue, which is not possible
e. Finally, find the error in the top part of Fig H1 explaining NIL formation.
You are never seeing homozygous blue parts because you always cross with a red parent
They forgo to mention one round of selfing to become partially homozygous blue, otherwise it
would not be possible to be homozygous blue in parts because you backcross with the red parent
(as we answered).

Note: You may wonder why put this figure in the manual? Well, when you get used to really studying
figures you will see such mistakes very often, even in high impact journals, and of course there is a
didactic reason ;-)
2.
a. What is the chance that a given marker (i.e. a difference between P1 and P2) has become
homozygous after 7 generations of selfing? 98.44% ~ 1-1/27=~99%
b. Explain why a CAPS marker is codominant and an AFLP marker is dominant (If you do not know
what CAPS/AFLP is, check Wikipedia). With CAPS you can distinguish heterozygosity and
with an AFLP you can only see if something is absent/present

c. Have a look at the pdf with the marker data. For which marker(s) in our experiment can we check
for homozygosity? Are all plants indeed homozygous as expected? P1 and P2 because they have
both restriction patterns. All the plants are homozygous because one has both patterns

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 Only for the CAPS marker (GAPB) you can check for homoyzugoisty as you need a co-
dominatn markers
All plants are homozygous for this particular markers
d. Arabidopsis plants in nature are largely homozygous, why is that?
Arabidopsis are self-fertilizing plants, so you would expect they would be homozygous
Arabidopsisi will mostly self in nature. As seen in previous questions selfing increases
homozygosity.

Now open the Excel file and fill it in further using the questions below as a guide:

The first tab “Marker Scores” lists the RILs and their alleles for the 6 markers.
- Score the alleles for the first 10 RILs (the rest has been done for you)
- Have a look at the recombinant analysis on the right, do you understand how the table is constructed?
Which genotypes are the recombinants?
- Now use the genotype numbers of the marker combinations from the table to make pair-wise
combinations of marker alleles in the tabs marked “M1 & M2”, “M1 & M3” etc.. Fill in the observed
totals per row/column. Do this for all combinations of markers 1-6 in the 63 plants (some have been
done for you) and answer the questions.
3. Is there equal segregation for the alleles of all markers (as expected from the first law of Mendel)? If
not, why do you think this happened? No, they’re not equally segregated as a result of linkage or
segregation distortion.

4. In the tabs you filled in, a Chi-square test is performed to determine linkage. Explain how the Chi-
square test can show linkage. Because it looks at the difference between the observed and the
expected values Why are the expected numbers not equal? The expected values are calculated
with the observed values
5. A significant deviation between observed and expected numbers of the four possible genotypes of a
gene pair may indicate linkage. Is in each case in which you find χ 2-value >3.84 the recombinant
fraction indeed lower than 50%? Which genes do you conclude to be linked? Fill in the two matrix
tables below here. In the first matrix indicate linkage between markers in the first matrix with a “+”
and in case of no detected linkage a “–“.
In the tabs also the (corrected) recombinant frequency RF has been calculated, using the formula RF =
p / (2-2p), in which p is the fraction of recombinants in the RILs ( # recombinants / # plants). This
correction has to be made because this is not a normal F2 population (Realise that RILs have
undergone multiple rounds of meiotic recombination during the selfings; for details and explanation of
the formula see Stam 2007). Fill in the RF values in the second matrix.

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Linkage or no linkage?

M1 (GAPD) M2 (m464) M3 (m269) M4 (m204) M5 (m162) M6 (m116)

M1 (GAPD)            
M2 (m464)  +          
M3 (m269)  - -         
M4 (m204)  +  + -       
M5 (m162)  -  -  + -     
M6 (m116)  -  +  -  + -   
+ means linkage
- means no linkage
+ = linkage group 1
+ = Linkage group 2

Fill in the RF values

M1 (GAPD) M2 (m464) M3 (m269) M4 (m204) M5 (m162) M6 (m116)
M1 (GAPD)            
M2 (m464)  0.23          
M3 (m269)  0.42 0.35         
M4 (m204)  0.13  0.11 0.33       
M5 (m162)  0.48  0.48  0.11 0.45     
M6 (m116)  0.43  0.13  0.40  0.29 0.55   

6. Use the first matrix table to determine what are the linkage groups. Marker 1 and 6 are not linked
but both marker 1 and 6 are linked to marker 2, explain how this is possible? Marker 2 is in between
of marker 1 and 6. 1 and 6 are too far away to be linked but are linked via 2.
Are markers 1 and 6 on the same linkage group? Yes
How many linkage groups are there, and which markers are in them? Two (see table)
7. Draw a genetic map with all six markers on the corresponding linkage group(s). using the data from
the matrix tables you filled in.
Linkage group 1: marker 1,2,4 & 6
Linkage group 1 marker 3 & 5

8. Next, calculate average values for the flowering time QTL for the A and B alleles for each marker.
(hint: use the integrated sort function in line 5 of the excel sheet to group the flowering times to A and
B). Can you already see which marker is most relevant for flowering time: or in other words, for which
marker does it matter most from which parent it is derived? Markers 3 and 5

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Now show the results in graph with the average flowering time on the y-axis and the markers on the x-
axis and A and B as categories. What can be concluded with respect to the position of the QTL? Try
to position the QTL on the linkage map.

The QTL is located on the linkage map of markers 3 and 5, but a more precise location cannot
be determined.
9. In fungi mapping populations can be quickly made by plating spores and phenotyping and
genotyping these. Explain why mapping populations in Arabidopsis take so much more time. The
plants must first be made homozygous which can take several generations

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

before you go home. After the feedback lecture please upload your finalised report.

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