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genetics_Lab_5_ sordaria - Download as PowerPoint by xb4Fd3

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									     Sordaria Lab
Determining Cross Over Frequency
                        Sordaria fimicola
•   Sordaria fimicola is an ascomycete fungus
•   Sordaria is a haploid (n) organism for most of
    its life cycle.
•   When the mycelium from two individuals
    meet, a diploid zygote (2n) is formed.
•    The diploid zygote then undergoes meiosis
    to yield 8 haploid ascospores. These
    ascospores exist in a narrow pod called an
    ascus (plural, asci). Many asci will grow
    together forming a reproductive structure
    called a perithecium.
•   When ascospores are mature the ascus
    ruptures, releasing the ascospores. Each
    ascospore can develop into a new haploid
Exchange of genetic material between homologous chromosomes
    Crossing-over in Sordaria sp.
      To observe crossing over in Sordaria:
make hybrids between wild-type & mutant strains

  Wild-type Sordaria:         Mutant strain:
  black spores (t+)           tan spores (t)
   Crossing-over in Sordaria sp.

When mycelia of these
two different strains
come together and
undergo meiosis, the
asci that develop will
contain four black
ascospores and four tan
Crossing-over in Sordaria sp.
               The arrangement of the
               spores directly reflects
               whether or not crossing
               over has occurred
     Today: Preparing the crosses
• If a strain producing tan spores is inoculated
  on one half of the plate and a strain
  producing black spores is placed on the
  other half, hyphae grow from both points
  and eventually meet at the center of the
  plate where they fuse in the equivalent of
• Since the hyphae of both strains are haploid,
  the fusion product is diploid. The diploid
  hyphae start to differentiate into a fruiting
  body called a perithecium as seen to the
  right. You can see the perithecia forming in
  the picture of the Petri plate; they are the
  dark line down the center of the plate.
         Today: Preparing the crosses
What we will attempt to do in this lab is to subject Sordaria fimicola to various
environmental conditions while crossing two strains.

 • Each table will be responsible
   for crossing two different strains
   of Sordaria: Tan X Wild

 • Each group at a table will be
   responsible for a specific
   crossing temperature: RT & 30oC
         Setting up the petri dish
• Divide an empty petri dish with crossing
  agar into four quadrants using a marker.
  Label each quadrant with the strain that
  will be placed there.
• Sterile techniques must be utilized at all
  times. Gently dip a spatula in alcohol and
  flame lightly to sterilize.
• Use the spatula to cut and remove a
  square of the growing Sordaria from the
  stock plate. Place the square in its
  respective location on the crossing plate.
• Parafilm the plates to seal them against
      How do I know when my Sordaria
           plate is ready to use?
Use a wooden toothpick to scrape a few perithecia from the plate periphery, where the 2
    strains have grown together.
Place the perithecia on a microscope slide, add a drop of water, and gently press down on
    them with a coverslip.
The perithecia should break open with gentle pressure and you should see asci with
    spores (they resemble bubblegum balls in a wrapper). You should also see a color
    difference between the 2 spore types in their crossover arrangement.

    Here are some signs the Sordaria asci are not ready to be used:
•   You use a lot of pressure to break open the perithecia (the black spheres).
•   The spores are greenish in color.
•   You only see darl, sac-like perithecia and no asci with spores.
•   If the culture is not ready, continue to incubate the plate at 25° C (77° F) and repeat
    the testing process until the asci mature and rupture easily on the slide. If the culture
    is still not ready on Friday, the plate can be refrigerated over the weekend. Allow the
    plate and culture to come to room temperature before rechecking on Monday.
     Procedure for counting the spores
1.    Use a scalpel to gently scrape the surface
     of the nutrient medium where the two
     strains intersect to collect perithecia. At
     the intersection of the two strains is the
     region to harvest the perithecia.

2.    Place the perithecia in a drop of water
     on a slide. Cover with a coverslip and
     return to your work area. Using the
     eraser end of a pencil (or a toothpick),
     press down the coverslip gently so that
     the perithecia rupture but the ascospores
     remain in the asci.

3.   View your slide using the 10X objective
     and locate a group of hybrid asci
                 Counting ascospores
•   Count at least 100 hybrid asci and enter your
    data in Table 1
•   Those on which the pattern of spore
    distribution in the ascus is 4 tan to 4 black
    were produced from cells in which no crossing
    over occurred. Such asci are called non-
•   Other asci contain black and tan spores that
    are distributed in 2:4:2 patterns or 2:2:2:2
    patterns. These asci only result from cells in
    which crossing over has occurred and are
    called recombinants. Because the
    recombinant patterns result only from
    crossing over, the frequency of occurrence of
    recombinants is a measure of how often
    crossing over occurs.

                         # of asci       # of asci                           Gene to
                       not showing       showing                           Centromere
                                                       Total   %MII Asci    Distance
                       crossing over   crossing over
                          MI Asci         MII Asci                           %MII/2

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