Lab 6 Continuation of Neurospora genetics

Objective:
1. Examine perithecia, asci and ascospores of Neurospora.
2. Try to pick random perithecia and ascospores under dissecting microscope.
3. Try to observe the protein localization under fluorescence microscope.
4. Get a rough image for different Basidiomycetes morphology.
The purpose for this exercise is to get a basic idea of Neurospora sexual life cycle and structure for different sexual reproduction stage.

Method:

• 2 week old Neurospora mating plate, find shot perithecium or perithecium on plate (black beads)
• Clush perithecium on slide, observe the ascospores released from perithecium.
• Transfer some free ascospores on water agar plate.
• Incubate for one week.
• Pick ascospores from the water agar plate to tubes.
• Incubate at 4 degree to get hydrated
• Heat shock at 65 degree for 45 min to kill contaminated conidia.
• Transfer the ascospores to medium with nutrition, ascospores should germinate.

Result:
1.Asci and ascospores are observed with clush perithecium.
Neurospora is heterothallic, it requires different mating type for sexual reproduction. Opposite mating type hyphae mate to produce perithecia. At maturity, many asci are developed within perithecia. Each ascus contains 8 ascospores. Under scope, there are intact ascus and ascospores released from broken asus. However,Pro-perithecium has no content.

2. Premature ascus with 8 ascospores.
Usually, after ascospores matured,ascospores are pigmentated. Four of them are dark color, the other four are light green, which perfectly reflect genetic linkage.

3. Observe SMRP10 mutant nuclei under fluorescence microspores.

One of the feature for Neurospora is it has multinuclei. In this mutant, histone protein which is the most abundant protein in nuclei is tagged with GFP. Histone protein can help to localize nuclei. 

4. Observe different Basidiomycete morphology.
The most conspicuous and familiar Basidiomycota are those that produce mushrooms, which are sexual reproductive structures. Basidiomycota have a huge impact on human affairs and ecosystem functioning. Many Basidiomycota obtain nutrition by decaying dead organic matter, including wood and leaf litter. Thus, Basidiomycota play a significant role in the carbon cycle. Unfortunately, Basidiomycota frequently attack the wood in buildings and other structures, which has negative economic consequences for humans. Humans have found diverse uses for Basidiomycota. Mushrooms, both cultivated and wild, are eaten in many countries. For the untrained, mushroom-hunting is a risky endeavor, because some Basidiomycota produce deadly toxins.

Discussion:
From this exercise, I get a rough idea of Neurospora sexual reproduction process. I learned the technique for mating and picking perithecium. This exercise teaches me some fundamental techniques to work with ascomycetes.

By observing morphology of different mushroom, I learn mushrooms belong to Basidiomycota phylum. The knowledge from textbook can correlate to our daily life. We should not taste wild mushroom if we are not 100% sure about the safty since many wild mushrooms produce toxin.