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Speciation  Lab 

Microbotryum pre-mating isolation

Background
Experiment
GenBank Assignment
Methods for Instructors
Instructor Hints

An alternative to the Phytophthora lab

Background:

Microbotryum spp. is a fungus that causes anther-smut disease in a large number of plant species (mostly in the carnation family - Caryophyllaceae - and related groups.)  Indeed in North America and Europe, anther smuts are known to occur on well over a hundred species from this family. Historically/traditionally all the anther-smuts have been put into a single species (Microbotryum violaceum). One question is whether they actually represent the same species and whether isolates from different hosts can interbreed.

 
Silene caroliniana with microbotryum
Silene caroliniana
with Microbotryum infection (center)
(photo Michael Hood)


Obtaining Microbotryum

For samples of strains contact Dr. Michael Hood - University of Virginia, Department of Biology, Charlottesville, VA 22904.
http://www.people.virginia.edu/~meh2s/tetradcollection.htm

Laboratory Exercise

We will count frequency of matings between the different strains of Microbotryum.

Strains are available of each mating type (A1 and A2) from each of the following host species

1. Silene alba - Mountain  Lake Biological Station, Virginia
2. Silene caroliniana  -  Kentucky
3. Silene dioica - Great Cumbrae Island, Scotland
4. Silene virginica - Duke Forest, North Carolina
5. Silene vulgaris - Alps, Switzerland
6. Saponaria officinalis  - Italy
7. Lychnis flos-cuculi  - Great Cumbrae Island, Scotland

Focal testers:
S. alba  A1, A2
S. caroliniana A1, A2

Crossing Design


 opposite mating type from tester        
Silene alba or Silene caroliniana A1 or A2
Silene alba

Silene caroliniana

Silene dioica

Silene vulgaris

Saponaria officinalis

Lychnis flos-cuculi



Data Collection


At 24 hr. (approx. -  but note time)
At 72 hr. (approx. - but note time)

GenBank assignment with Microbotryum



Methods - For Instructors

wedge image


Sample Class Data (UVA 2002)

The following class we  combined everyone's data on the board and calculated averages. These are our results as % sporidia mating.

HOST NAME
Mating Type
S. alba      
S. dioica
S. vulgaris
S. virginica
S. caroliniana
Sap. officialis
L. flos-cuculi
S. alba
A1
58
39
32
45
31
17
28
S. alba
A2
39
23
24
32
36
12
25
S. caroliniana
A1
19
21
15
22
36
21
24
S. caroliniana
A2
22
20
19
30
41
21
18

Hints on GenBank Exercise

The students then were asked to compare these data with two types of distance matrices, geographical and phylogenetic. Given the localities for each sample, they used maps or the web to determine geographical distances. They used NCBI's BLAST search for ITS sequences in GenBank to determine genetic relatedness (this was explained as a very rough estimate of genetic relatedness and we did some more rigorous phylogenetic work later in the semester)

One change that is worth suggesting is the use of M.v.- Silene virginica instead of M.v. -Silene caroliniana because the ITS sequence for M.v. Silene caroliniana is not found in GenBank. Also, it should be noted that the samples from S. alba at Mountain Lake are actually native to Europe, whereas the samples from S. virginica are native to North America.

We gave the students readings that were the same as for the Phytophthora lab, with the addition of Antonovics et al. (1996) American Midland Naturalist 135:130-143. The students were also given a brief introduction to the anther-smut disease system using images from our webpage http://www.people.virginia.edu/~meh2s/tetradcollection.htm .

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