FUNGAL POPULATION LAB
Overview:
In this exercise, I’m going to ask you to take DNA sequence data, determine if
collections from different geographical areas have sequence differences, identify
restriction site differences and use these restriction site differences to tell me something
about the population(s).
Some Background:
Eukaryotic Ribosomes are composed of two subunits and each subunit contains
ribosomal RNAs and a number of proteins. The ribosomal RNAs (rRNAs) are
transcribed together then processed to produce the separate rRNAs (Figure 1). The
ribosomal RNA transcription block is repeated many times as a tandem repeat in the cell.
All of the repeats are usually identical but the mechanism for “homogenization” is
unknown.. Prokaryote rRNAs are smaller and do not exist as a tandem repeat. The
eukaryotic 18S (Small ribosomal ) rRNA, 5.8S rRNA and ca. 27S (or large subunit
ribosomal) RNAs are separated by transcribed spacers called internally transcribed spacer
1 (ITS1) and internally transcribed spacer 2 (ITS2). The region from the 3’ end of the
18S rRNA gene through the 5’ end of the LSU rRNA gene is variable at the species and
sometimes population level and can be used to identify species, dissect species
relationships and determine population structure. In this exercise, we will use this region
to answer questions about the population structure of the common coral fungus
Artomyces pyxidatus (correct names but most fungal books list it as Clavicorona
pyxidata)
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Exercise 1. Determine sequence differences.
Open the file Fungallab.msf with any text editor including Word. [An msf file is an
aligned sequence file produced by the Genetics Cooperative Group programs (GCG).
GCG is available for our use on the University’s Unix computer “Larry” but you must
have a Unix account to use it. These accounts are free to you as a student.] Print out a
copy of the file.
Examine the aligned sequences from different geographical regions and identify the
differences in sequence that might be used to evaluate geographical structure. Mark
these on your copy.
Answer the following questions: Are there any differences between sequences from
collections within the North and Central American collections? European and North
American collections. What geographical regions show the most difference? Propose a
hypothesis to suggest why this might be so.
Yes, there are a few differences … around 160 and 440, 495.
Between European and north American collection we have 101,
151, 500, 551, 601. The greatest difference seems to be
between the European groups and the American groups (north
and central). This could be because of historical patterns
of dispersal.
Exercise 2: Determine restriction site differences in North American
collections
Open the file Fungallab_fasta.rsf. This file presents the same data as the msf file but now
each sequence is given separately and gaps in the sequence induced for alignment
purposes have been removed. You will determine restriction sites for one European
collection, one collection from Mexico and one collection from northeastern USA.
Go to http://tools.neb.com/NEBcutter2/index.php
This site contains a handy tool to map restriction sites for any given DNA sequence. Cut
and paste the DNA sequence for one of the sequences in the Fungallab_fasta.rsf list.
Select “all commercially available enzymes” and run the program. The program will
display restriction sites for the entire sequence. You can mark a specific area of the
sequence and zoom in on that area using options given on the results page.
Locate regions in which there are differences between the North American collections
and type in that section of DNA for one of the collections. Make sure to delete any gaps.
Run the program and print the results for reference. Repeat this with the second
collection.
Answer the following questions:
2-1. What is a restriction enzyme?
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An enzyme that cuts a DNA sequence of a certain code.
2-2.
What restriction enzymes cut a sequence from Mexico/Costa Rica but don’t cut a
sequence from eastern NA?
BSAJ1 cuts mexico but not NA .
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
2-3.
What restriction enzymes cut a sequence from Eastern North America but not
Mexico/Costa Rica?
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QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
In using Taq1, we can cut the sequence of new york, TCGAGGTGAA, but there is no
restriction enzyme for the maine cut, at TCAAGGTGAA, so we can differentiate
between populations.
Go to http://rebase.neb.com/rebase/rebhelp.html
Search rebase for the enzymes you have identified as distinguishing between collections
from different geographical regions.
2-4. What is the recognition sequence for each of the enzymes?
Exercise 3: Determine the restriction fragment length polymorphisms
(RFLPs) that you would see on a gel if you cut the ITS1-5.8S-ITS2 region
with each enzyme you have identified.
Go to: http://arbl.cvmbs.colostate.edu/molkit/mapper/index.html
Copy and paste one of your ungapped sequences and click on “create map”. In the lower
box, you will see a map by enzyme. Estimate fragment sizes on a gel from the data for
each of the enzymes you have identified. Draw the patterns you would expect to see on
a gel below. Repeat the process with your other sequence.
3-1. Can you identify the different sequences (haplotypes) by gel electrophoresis? How?
Exercise 4: Mapping geographical patterns
Open the file restriction pyx-haplotypes.rft in any text file editor including word. This
file gives the restriction pattern for a number of collections. Map these collections on
the map template attached.
4-1: Is there any geographical signal present or is this one large homogeneous
population?
4-2: How do you explain discrepancies in the RFLP patterns such as 0,0 and 1,1
restriction patterns? What may be causing these?
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4-3: If you consider North America as one large interbreeding population, is the
population in Hardy Weinberg equilibrium? Calculate the haplotype frequencies and
estimate the frequency of p2, 2pq, q2. How do these compare with observed frequencies.
4-4: What do you conclude about North American Artomyces pyxidatus Propose
hypotheses that might explain your data.
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