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Tetrad Analysis


Random strand analysis

•  So far we have been doing gene mapping using what is known as random strand analysis. With this method, you capture each gamete one at a time and count it as recombinant or nonrecombinant.

•  But each gamete is only one of the four cells produced by a single meiosis. You only see 1/4 of what happened during that meiosis.

•  With Drosophila (and most other organisms) you can only do random strand analysis.

•  Because each gamete contains only one of the four chromatids from the bivalent, you get incomplete information about what happened during meiosis.

•  What if you could capture all four cells (a tetrad) from a single meiosis as a unit?

Tetrad analysis

•  With tetrad analysis, you get more information about the crossing over process and linkage because you capture a whole tetrad.

•  This enables you to determine how recombination took place (or didn't) for the entire bivalent.

•  So random strand analysis is "chromatid analysis"; but tetrad analysis is "bivalent analysis."

•  So if we can't use Drosophila for this, what kind of organism would work?

•  We need an organism which would allow a way to catch all four meiotic products and know that they came from the same mother cell.

•  The best organisms for this are sac fungi (ascomycetes) such as yeast or Neurospora (pink bread mold).

•  The products of meiosis are ascospores, which remain in a sac (the ascus) long enough to be analyzed as a unit.

More detail about crossing over

•  A single crossover only involves two of the four chromatids in a bivalent.

Crossing over
Result of single crossover in non-sister chromatids.
Pierce, Genetics: A Conceptual Approach, W.H. Freeman and Company

•  With random strand analysis, you can only tell if a single chromatid is recombinant or nonrecombinant.

•  To see that that both recombinant and nonrecombinant chromatids can occur at the same time, we must use tetrad analysis.

•  Crossing over (one chiasma) always involves non-sister chromatids.

•  Bivalent in salamander oocyte. (arrows indicate chiasmata)

Chiasmata
Chiasmata in diplotene bivalent of salamander oocyte. Kinetochores are visible as darkly stained circles; arrows point to chiasmata.
Micrograph courtesy of James Kezer

Using Neurospora for tetrad analysis

•  Neurospora 's life cycle is based on zygotic meiosis

Ascomycete Life Cycle
Life cycle of typical ascomycete fungus, such as Neurospora.

•  The zygote is the only diploid cell in the life cycle

•  So all other cells are haploid

•  Tetrad analysis is also called "haploid mapping."

•  Since cells are haploid, there is only one copy of each allele, so alleles are always expressed. This means you don't have to worry about whether an allele is dominant or recessive.

•  An ascus contains all the products of a single meiosis.

•  After meiosis, there is one mitosis division, so the mature ascus has 8 ascospores, but these are really four pairs of "twins."

•  Spores can be removed from the ascus with a micro-manipulator, grown into separate cultures, and analyzed to find their phenotypes.

Centromere mapping

•  Because Neurospora's ascus is linear, the spores remain in the order they were formed. They can't move around within the ascus.

•  So the order of the ascospores actually reflects the 2 divisions of meiosis.

•  This fact means that you can detect crossing over between a single locus and the centromere and map this distance.

•  How the spore order reflects whether or not a cross over has occurred between a locus and centromere.

•  First division segregation

First Division Segregation
Spore patterns in ascus for first division segregation.
Drawing by John Tiftickjian

•  No recombination between gene and centromere

•  The 4 spores in each half of the ascus are the same.

•  It's called a first division segregation because after meiosis I, each daughter cell has either D or d, but not both. D and d have segregated to different cells after one cell division.

•  Second division segregation

Second Division Segregation
Spore pattern in ascus for second division segregation.
Drawing by John Tiftickjian

•  Crossing over has happened between gene and centromere

•  The 4 spores in each half of the ascus are not all the same.

•  It's called a second division segregation because after meiosis I, each daughter cell has both D and d. D and d do not segregate to different cells until meiosis II.

•  Neurospora cross results showing first and second division segregation patterns

Neurospora asci
Asci of Neurospora resulting from cross between strains with different spore colors.
Dr. N. B. Raju, Department of Biological Sciences, Stanford University

•  Example cross for centromere mapping

Tetrad analysis cross

•  Add numbers of 2nd division asci (6 + 5 + 6 + 7 = 24)

•  Divide this number by the total number of asci (24/100 = 0.24)

•  Convert to a percentage (0.24 x 100% = 24). This is the percentage of asci that resulted from meiosis in which there was a crossover between the gene locus and the centromere.

•  Divide this number by 2 to get the map distance (24/2 = 12 mu). You must do this step because map units are based on the percentage of chromatids that are recombinants. For each ascus with a single crossover, only two of the four chromatids are recombinants.

Tetrad analysis can show exactly which chromatids are involved in double crossovers using three-point crosses.

Double crossover types

•  Note that when a double crossover happens, the two crossovers my involve only 2 chromatids, 3 chromatids, or all 4 chromatids.

•  You can detect which chromatids have done crossovers by seeing which kinds of recombinant spores are present together in the same ascus.

•  In random strand analysis, you can only tell if a single chromatid has had multiple cross-overs, but you can't tell what the other two chromatids of the bivalent did in the same meiosis.

More on Neurospora

•  Lots of additional information on using Neurospora in genetic analysis and for studies of meiosis can be found on the web at the Perkins Laboratory at Stanford University

Visit The Perkins Laboratory on the web

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