Preparing Maize Synaptonemal Complex Spreads and Sequential Immunofluorescence and Fluorescence In Situ Hybridization
Immunofluorescence and fluorescence in situ hybridization (FISH) can be used to locate specific proteins and DNA sequences, respectively, in chromosomes by light microscopy. Here we describe sequential use of these techniques on spreads of maize synaptonemal complexes (SCs) to determine whether crossing over can occur in knob heterochromatin. We used antibodies to AFD1, an SC protein, and MLH1, a class I (interference-sensitive) crossover protein found in most recombination nodules (RNs) to identify crossovers (COs) along SCs. Next, we used FISH to localize a 180 bp knob-specific tandem repeat. Combining immunofluorescence and FISH images of the same SC spreads showed that heterochromatic knobs do not prohibit class I COs. This technique is broadly applicable to investigations of plant prophase I chromosomes where meiotic recombination takes place.
Key wordsAFD1 Crossing over Fluorescence in situ hybridization Heterochromatin Immunofluorescence Knobs Maize MLH1 Recombination nodules Synaptonemal complex
We thank the National Science Foundation for supporting our work and Associate Professor Song-Bin Chang in the Department of Life Sciences at National Cheng Kung University, Taiwan for selecting the PCR priming sequences for knob DNA amplification.
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