The overall plan involves 9 steps divided between the University of Houston and Baylor College of Medicine. The process is iterative, and Steps 3-8 will initially occur as a series of 5 overlapping events. Refinement of the map involves repeating Steps 6-8 for additional F2 individuals. The genetic map will be confirmed by Fluorescence in situ Hybridization (FISH) to identify the physical location of a subset of markers on individual chromosome.

• Step1: Identify unique Simple Sequence Repeats (SSRs) in X.tropicalis genome sequence (JGI), and generate PCR primers to amplify these SSRs.
• Step2: Generate Map cross DNA panel from >500 F2 individuals.
• Step3: Test the amplification of primers synthesized from Step 1.
• Step4: Determine which amplifiable SSRs are polymorphic in our samples.
• Step5: Synthesize fluorescent primers for those SSRs that are polymorphic.
• Step6: Perform multiplex PCR reactions using fluorescent primers and the map cross DNAs.
• Step7: Analyze PCR reactions on ABI 3730XL and collect output data.
• Step8: Utilize output data and assemble a genetic map using the MAPMAKER program.
• Step9: Physical confirmation of linkage map via FISH.