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jmodlis edited this page Aug 25, 2017 · 21 revisions

TetradAnalysis

Overview

This program is intended to be used as a tool to aid in counting pollen tetrads in three color fluorescent intervals (fluorescent tagged lines, or FTLs). The user is expected to either have 1) an image datafile in three colors (e.g., CFP, YFP, and dsRED) or 2) a slide with three-color pollen tetrads on an epifluorescence microscope.

For the original description of the FTLs, see here:

Francis KE, Lam SY, Harrison BD, Bey AL, Berchowitz LE and Copenhaver GP (2007) Pollen Tetrad-based Visual Assay for Meiotic Recombination in Arabidopsis. Proc. Natl. Acad. Sci.104: 3913-3918. doi:10.1073/pnas.0608936104

For a description of the different tetrad types, see here:

Berchowitz LE, Copenhaver GP (2008) Fluorescent Arabidopsis tetrads: A visual assay for quickly developing large crossover and crossover interference datasets. Nature Protocols 3(1): 41-50. doi:10.1038/nprot.2007.491

Using the program

  1. After clicking on the jar file, the gui will appear. The program allows the user to record the tetrad type of each tetrad viewed, creating a raw data file of tetrad types and other sample information for each tetrad that was analyzed.

  2. The filename is the name of the output file, which will be written to the same directory that the jar file is in.

  3. Date, sample name, temperature, and tetrad number (start at 1) should be filled in.

  4. For the FTL marker order, indicate the order of fluorescent proteins on the chromosome, going from the left to right (or top to bottom) on the chromosome. It is very important that this order is correct -- without this information, the program will not output the correct tetrad type.

  5. For the tetrad type, the user should either chose a tetrad type from the drop-down box or click on the boxes to indicate which pollen grains are fluorescing for each fluorescent protein. If the user choses something other than "none selected" from the drop-down box, the program will default to what is chosen in the drop down box over the tetrad configuration grid. The grid is useful for more complicated configurations.

  6. When all of the information is filled out for a single tetrad, the user should click on the "Record Data Now!!!" button. This will write the results to the output file. The tetrad number will be incremented by 1 and all information will remain in the text fields.

  7. The "Reset sample fields" button will clear all fields except for the filename. FTL marker order will remain as set by the user.

Screen shot of gui

Output

The program outputs a text file of the raw data (specified by filename field), with the following information on each line:

  1. Date - useful if the user would like to analyze data from different days separately.
  2. Sample name - should be unique for each sample.
  3. Temperature - the temperature in which the plants were grown. Under normal conditions, this parameter will not vary.
  4. Tetrad number - this number is useful for record-keeping when image files for each tetrad/sample are available.
  5. Tetrad type (A-L) - these tetrad types are explained in Berchowitz et al. 2008 (see above).
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